Ebola peptides and immunogenic compositions containing same

ABSTRACT

Using CTL epitopes to the Ebola GP, NP, VP24, VP30, VP35 and VP40 virion proteins, a method and composition for use in inducing an immune response which is protective against infection with Ebola virus is described.

[0001] This application is a continuation-in-part of U.S. utilityapplication Ser. No. 09/337,946, filed Jun. 22, 1999, which claimspriority from U.S. provisional application No. 60/091,403 (filed Jun.29, 1998). The entire contents of both applications are incorporatedherein by reference.

BACKGROUND OF THE INVENTION

[0002] Ebola viruses, members of the family Filoviridae, are associatedwith outbreaks of highly lethal hemorrhagic fever in humans and nonhumanprimates. The natural reservoir of the virus is unknown and therecurrently are no available vaccines or effective therapeutic treatmentsfor filovirus infections. The genome of Ebola virus consists of a singlestrand of negative sense RNA that is approximately 19 kb in length. ThisRNA contains seven sequentially arranged genes that produce 8 mRNAs uponinfection (FIG. 1). Ebola virions, like virions of other filoviruses,contain seven proteins: a surface glycoprotein (GP), a nucleoprotein(NP), four virion structural proteins (VP40, VP35, VP30, and VP24), andan RNA-dependent RNA polymerase (L) (Feldmann et al.(1992) Virus Res.24, 1-19; Sanchez et al.,(1993) Virus Res. 29, 215-240; reviewed inPeters et al. (1996) In Fields Virology, Third ed. pp. 1161-1176.Fields, B. N., Knipe, D. M., Howley, P. M., et al. eds. Lippincott-RavenPublishers, Philadelphia). The glycoprotein of Ebola virus is unusual inthat it is encoded in two open reading frames. Transcriptional editingis needed to express the transmembrane form that is incorporated intothe virion (Sanchez et al. (1996) Proc. Natl. Acad. Sci. USA 93,3602-3607; Volchkov et al, (1995) Virology 214, 421-430). The uneditedform produces a nonstructural secreted glycoprotein (sGP) that issynthesized in large amounts early during the course of infection.Little is known about the biological functions of these proteins and itis not known which antigens significantly contribute to protection andshould therefore be used to induce an immune response.

[0003] Recent studies using rodent models to evaluate subunit vaccinesfor Ebola virus infection using recombinant vaccinia virus encodingEbola virus GP (Gilligan et al., (1997) In Vaccines 97, pp. 87-92. ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y.), or naked DNAconstructs expressing either GP or sGP (Xu et al. (1998) Nature Med. 4,37-42) have demonstrated the protective efficacy of Ebola virus GP inguinea pigs. (All documents cited herein supra and infra are herebyincorporated in their entirety by reference thereto.) Additionally,Ebola virus NP and GP genes expressed from naked DNA vaccines(Vanderzanden et al.,(1998) Virology 246, 134-144) have elicitedprotective immunity in BALB/c mice. There has been one study that showedprotection in nonhuman primates with a high dose DNA prime/high doseadenovirus boost and a 6 pfu challenge. However, this study provideslimited benefit for humans or non-human primates because such highdosing is unlikely to be given to humans due to high inherent risks andother factors. So there still exists a need for a human vaccine which isefficacious for protection from Ebola virus infection.

SUMMARY OF THE INVENTION

[0004] The present invention satisfies the need discussed above. Thepresent invention relates to a method and composition for use ininducing an immune response which is protective against infection withEbola virus.

[0005] Because the biological functions of the individual Ebola virusproteins were not previously known and the immune mechanisms necessaryfor preventing and clearing Ebola virus infection were not previouslywell understood, it was not known which antigens significantlycontribute to protection and should therefore be included in an eventualvaccine candidate to induce a protective immune response. However, theinventors have induced protection against Ebola infection in mammalsusing virus replicon particles (VRPs) expressing the Ebola GP, NP, VP24,VP30, VP35 or VP40 genes. These VRPs and some uses are described inco-pending application Ser. No. 09/337,946 (filed Jun. 22, 1999), theentire contents of which are hereby incorporated by reference.

[0006] One embodiment of the present invention entails a DNA fragmentencoding each of the Ebola Zaire 1976 GP, NP, VP24, VP30, VP35, and VP40virion proteins (SEQUENCE ID NOS. 1-7).

[0007] Another embodiment provides the DNA fragments of Ebola virionproteins in a recombinant vector. When the vector is an expressionvector, the Ebola virion proteins GP, NP, VP24, VP30, VP35, and VP40 areproduced. It is preferred that the vector is an alphavirus repliconvector, especially a replicon vector that has the ability to produce thedesired protein or peptide in a manner that induces protective B and Tcells in vivo in mammals. Any alphavirus vector may be effective,including but not limited to the Venezuelan Equine Encephalitis (VEE)virus, eastern equine encephalitis, western equine encephalitis, Semlikiforest and Sindbis. For instance, in a preferred embodiment the VEEreplicon vector comprises a VEE virus replicon and a DNA fragmentencoding any of the Ebola Zaire 1976 (Mayinga isolate) GP, NP, VP24,VP30, VP35, or VP40 proteins. In another preferred embodiment, the VEEreplicon vector comprises a VEE virus replicon and a DNA fragmentencoding any of the amino acid sequences set forth in SEQ ID NOs:24-53.The construct can be used as a nucleic acid vaccine or for theproduction of self replicating RNA. To that end, a self replicating RNAof this invention can comprise the VEE virus replicon and any of theEbola Zaire 1976 (Mayinga isolate) RNAs encoding the GP, NP, VP24, VP30,VP35, and VP40 proteins described above, or the amino acid sequences setforth in SEQ ID NOs:24-53. The RNA can be used as a vaccine forprotection from Ebola infection. When the RNA is packaged, a VEE virusreplicon particle is produced.

[0008] Another embodiment entails infectious VEE virus repliconparticles produced from the VEE virus replicon RNAs described above.

[0009] Another embodiment of the invention encompasses peptides thatmake up cytotoxic T lymphocyte (CTL) epitopes corresponding to Ebola GP,NP, VP24, VP30, VP35, or VP40 proteins. The epitopes may include thesequences identified as SEQ ID NOS:24-53, as described below. A relatedaspect of this embodiment provides DNA fragments that respectivelyencode these Ebola peptides. A further embodiment relates to recombinantDNA constructs that express these epitope peptides.

[0010] An additional embodiment includes a pharmaceutical compositionthat includes one or more of these CTL epitope peptides (and preferablyone or more of SEQ ID NOs:24-53), in an effective immunogenic amount ina pharmaceutically acceptable carrier and/or adjuvant.

[0011] A further embodiment entails an immunological composition for theprotection of mammals including humans against Ebola virus infection,comprising at least one (but preferably at least two, and morepreferably at least three, and most preferably all) of the Ebola virusGP, NP, VP24, VP30, VP35, or VP40 proteins. In a related embodiment, thecomposition may include one or more of the CTL epitopes set forth in SEQID NOs: 24-53 (described below).

[0012] In a related preferred embodiment, the immunological compositionscomprise alphavirus replicon particles (such as, for instance, VEE virusreplicon particles) expressing the Ebola virus GP, NP, VP24, VP30, VP35,or VP40 proteins, or any combination of different VEE virus repliconseach expressing one or more different Ebola proteins selected from GP,NP, VP24, VP30, VP35 and VP40. For instance, in a preferred embodimentthe composition may include one or more of SEQ ID NOs: 24-53 (describedbelow). In another preferred embodiment, the composition includes atleast the VP30, VP35 and VP40 proteins.

[0013] An additional embodiment includes vaccines against infection byEbola, comprising virus replicon particles (preferably VEE virusreplicon particles) expressing the Ebola virus GP, NP, VP24, VP30, VP35,or VP40 proteins, or any combination of different VEE virus repliconseach expressing one or more different Ebola proteins selected from GP,NP, VP24, VP30, VP35 and VP40. For instance, in a preferred embodimentthe Ebola VRPs contain one or more of the peptides specified by SEQ IDNOs: 24-53. In a related embodiment, the vaccine may include at aminimum at least one of the Ebola proteins selected from GP, NP, VP24,VP30, VP35 and VP40. For instance, in a preferred embodiment the vaccineincludes at least the VP30, VP35 and VP40 proteins. In another preferredembodiment, the vaccine may include one or more of SEQ ID NOs: 24-53.

[0014] The invention also contemplates methods for inducing in a mammala cytotoxic T lymphocyte response to the Ebola virus GP, NP, VP24, VP30,VP35, or VP40 proteins, or to a peptide comprising at least 6 aminoacids thereof. In one version of the method, a recombinant DNA constructis administered to a mammal, such as, for example, a mouse, a guineapig, a monkey or a human, which a recombinant DNA construct expressesthe amino acid sequence of at least one of the Ebola virus GP, NP, VP24,VP30, VP35, or VP40 proteins (or a peptide comprising at least 6 aminoacids thereof), under such conditions that a protective CTL response isinduced in that mammal. In particular, the administered peptides mayinclude one or more of SEQ ID NOs: 24-53. In another version of themethod, one of the above-described immunogenic compositions isadministered to the mammal, and preferably one that comprises virusreplicon particles containing one of the Ebola virus GP, NP, VP24, VP30,VP35, or VP40 proteins (or a peptide comprising at least 6 amino acidsthereof), or including one of the CTL epitopes set forth in SEQ IDNOs:24-53. In another version of the method, the amino acid sequence ofat least one of the Ebola virus GP, NP, VP24, VP30, VP35, or VP40proteins (or a peptide comprising at least 6 amino acids thereof) isadministered to a mammal, such as, for example, a mouse, a guinea pig, amonkey or a human, under such conditions that a protective CTL responseis induced in that mammal. In particular, the administered peptides mayinclude one or more of SEQ ID NOs: 24-53.

BRIEF DESCRIPTION OF THE DRAWINGS

[0015] These and other features, aspects, and advantages of the presentinvention will become better understood with reference to the followingdescription and appended claims, and accompanying drawings where:

[0016]FIG. 1 is a schematic description of the organization of the Ebolavirus genome.

[0017]FIGS. 2A, 2B and 2C are schematic representations of the VEEreplicon constructs containing Ebola genes.

[0018]FIG. 3 shows the generation of VEE viral-like particles containingEbola genes.

[0019]FIG. 4 is an immunoprecipitation of Ebola proteins produced fromreplicon constructs.

DETAILED DESCRIPTION

[0020] In the description that follows, a number of terms used inrecombinant DNA, virology and immunology are extensively utilized. Inorder to provide a clearer and consistent understanding of thespecification and claims, including the scope to be given such terms,the following definitions are provided.

[0021] Filoviruses. The filoviruses (e.g. Ebola Zaire 1976) cause acutehemorrhagic fever characterized by high mortality. Humans can contractfiloviruses by infection in endemic regions, by contact with importedprimates, and by performing scientific research with the virus. However,there currently are no available vaccines or effective therapeutictreatments for filovirus infection in humans. The virions of filovirusescontain seven proteins: a membrane-anchored glycoprotein (GP), anucleoprotein (NP), an RNA-dependent RNA polymerase (L), and four virionstructural proteins (VP24, VP30, VP35, and VP40). Little is known aboutthe biological functions of these proteins and it is not known whichantigens significantly contribute to protection and should therefore beused in an eventual vaccine candidate.

[0022] Replicon. A replicon is equivalent to a full-length virus fromwhich all of the viral structural proteins have been deleted. A multiplecloning site can be inserted downstream of the 26S promoter into thesite previously occupied by the structural protein genes. Virtually anyheterologous gene may be inserted into this cloning site. The RNA thatis transcribed from the replicon is capable of replicating andexpressing viral proteins in a manner that is similar to that seen withthe full-length infectious virus clone. However, in lieu of the viralstructural proteins, the heterologous antigen is expressed from the 26Spromoter in the replicon. This system does not yield any progeny virusparticles because there are no viral structural proteins available topackage the RNA into particles.

[0023] Particles which appear structurally identical to virus particlescan be produced by supplying structural protein RNAs in trans forpackaging of the replicon RNA. This is typically done with two defectivehelper RNAs which encode the structural proteins. One helper consists ofa full length infectious clone from which the nonstructural proteingenes and the glycoprotein genes are deleted. This helper retains onlythe terminal nucleotide sequences, the promoter for subgenomic mRNAtranscription and the sequences for the viral nucleocapsid protein. Thesecond helper is identical to the first except that the nucleocapsidgene is deleted and only the glycoprotein genes are retained. The helperRNAs are transcribed in vitro and are co-transfected with replicon RNA.Because the replicon RNA retains the sequences for packaging by thenucleocapsid protein, and because the helpers lack these sequences, onlythe replicon RNA is packaged by the viral structural proteins. Thepackaged replicon particles are released from the host cell and can thenbe purified and inoculated into animals. The packaged replicon particleswill have a tropism similar to the parent virus. The packaged repliconparticles will infect cells and initiate a single round of replication,resulting in the expression of only the virus nonstructural proteins andthe product of the heterologous gene that was cloned in the place of thevirus structural proteins. In the absence of RNA encoding the virusstructural proteins, no progeny virus particles can be produced from thecells infected by packaged replicon particles.

[0024] Any alphavirus replicon may be effective in this invention, aslong as it has the ability to produce the desired protein or peptide ina manner that induces protective B and T cells in vivo in mammals towhich it is administered to (such as, for instance, eastern equineencephalitis, western equine encephalitis, Semlike forest, Sindbis andVenezualen Equine Encephalitis).

[0025] The VEE virus replicon (Vrep) is a preferred vector system. TheVrep is a genetically reorganized version of the VEE virus genome inwhich the structural protein genes are replaced with a gene from animmunogen of interest, such as the Ebola virus virion proteins. Thisreplicon can be transcribed to produce a self-replicating RNA that canbe packaged into infectious particles using defective helper RNAs thatencode the glycoprotein and capsid proteins of the VEE virus. Since thepackaged replicons do not encode the structural proteins, they areincapable of spreading to new cells and therefore undergo a singleabortive round of replication in which large amounts of the insertedimmunogen are made in the infected cells. The VEE virus replicon systemis described in U.S. patent to Johnston et al., U.S. Pat. No. 5,792,462issued on Aug. 11, 1998.

[0026] Subject. Includes both human, animal, e.g., horse, donkey, pig,mouse, hamster, monkey, chicken, and insect such as mosquito.

[0027] In one embodiment, the present invention relates to DNA fragmentswhich encode any of the Ebola Zaire 1976 (Mayinga isolate) GP, NP, VP24,VP30, VP35, and VP40 proteins. The GP and NP genes of Ebola Zaire werepreviously sequenced by Sanchez et al. (1993, supra) and have beendeposited in GenBank (accession number L11365). A plasmid encoding theVEE replicon vector containing a unique ClaI site downstream from the26S promoter was described previously (Davis, N. L. et al., (1996) J.Virol. 70, 3781-3787; Pushko, P. et al. (1997) Virology 239, 389-401).The Ebola GP and NP genes from the Ebola Zaire 1976 virus were derivedfrom PS64- and PGEM3ZF(−)-based plasmids (Sanchez, A. et al. (1989)Virology 170, 81-91; Sanchez, A. et al. (1993) Virus Res. 29, 215-240).From these plasmids, the BamHI-EcoRI (2.3 kb) and BamHI-KpnI (2.4 kb)fragments containing the NP and GP genes, respectively, were subclonedinto a shuttle vector that had been digested with BamHI and EcoRI (Daviset al. (1996) supra; Grieder, F. B. et al. (1995) Virology 206,994-1006). For cloning of the GP gene, overhanging ends produced by KpnI(in the GP fragment) and EcoRI (in the shuttle vector) were made bluntby incubation with T4 DNA polymerase according to methods known in theart. From the shuttle vector, GP or NP genes were subcloned asClaI-fragments into the ClaI site of the replicon clone, resulting inplasmids encoding the GP or NP genes in place of the VEE structuralprotein genes downstream from the VEE 26S promoter.

[0028] The VP genes of Ebola Zaire were previously sequenced by Sanchezet al. (1993, supra) and have been deposited in GenBank (accessionnumber L11365). The VP genes of Ebola used in the present invention werecloned by reverse transcription of RNA from Ebola-infected Vero E6 cellsand subsequent amplification of viral cDNAs using the polymerase chainreaction. First strand synthesis was primed with oligo dT (LifeTechnologies). Second strand synthesis and subsequent amplification ofviral cDNAs were performed with gene-specific primers (SEQ ID NOS:8-16).The primer sequences were derived from the GenBank deposited sequencesand were designed to contain a ClaI restriction site for cloning theamplified VP genes into the ClaI site of the replicon vector. Theletters and numbers in bold print indicate Ebola gene sequences in theprimers and the corresponding location numbers based on the GenBankdeposited sequences. VP24: (1) forward primer is (SEQ ID NO:8)5′-GGGATCGATCTCCAGACACCAAGCAAGACC-3′         (10,311-10,331)       (2)reverse primer is (SEQ ID NO:9) 5′-GGGATCGATGAGTCAGCATATATGAGTTAGCTC-3′      (11,122-11,145) VP30: (1) forward primer is SEQ ID NO:10)5′-CCCATCGATCAGATCTGCGAACCGGTAGAG-3′      (8408-8430)       (2) reverseprimer is (SEQ ID NO:11) 5′-CCCATCGATGTACCCTCATCAGACCATGAGC-3′        (9347-9368) VP35: (1) forward primer is (SEQ ID NO:12)5′-GGGATCGATAGAAAAGCTGGTCTAACAAGATGA-3′     (3110-3133)       (2)reverse primer is (SEQ ID NO:13)5′-CCCATCGATCTCACAAGTGTATCATTAATGTAACGT-3′         (4218-4244) VP40: (1)forward primer is (SEQ ID NO:14) 5′-CCCATCGATCCTACCTCGGCTGAGAGAGTG-3′           (4408-4428)       (2) reverse primer is (SEQ ID NO:15)5′-CCCATCGATATGTTATGCACTATCCCTGAGAAG-3′            (5495-5518) VP30 #2:      (1) forward primer as for VP30 above       (2) reverse primer is(SEQ ID NO:16) 5′-CCCATCGATCTGTTAGGGTTGTATCATACC-3′

[0029] The Ebola virus genes cloned into the VEE replicon weresequenced. Changes in the DNA sequence relative to the sequencepublished by Sanchez et al. (1993) are described relative to thenucleotide (nt) sequence number from GenBank (accession number L11365).

[0030] The nucleotide sequence we obtained for Ebola virus GP (SEQ IDNO:1) differed from the GenBank sequence by a transition from A to G atnt 8023. This resulted in a change in the amino acid sequence from Ileto Val at position 662 (SEQ ID NO: 17).

[0031] The nucleotide sequence we obtained for Ebola virus NP (SEQ IDNO:2) differed from the GenBank sequence at the following 4 positions:insertion of a C residue between nt 973 and 974, deletion of a G residueat nt 979, transition from C to T at nt 1307, and a transversion from Ato C at nt 2745. These changes resulted in a change in the proteinsequence from Arg to Glu at position 170 and a change from Leu to Phe atposition 280 (SEQ ID NO: 18).

[0032] The Ebola virus VP24 nucleotide sequence (SEQ ID NO:3) differedfrom the GenBank sequence at 6 positions, resulting in 3 nonconservativechanges in the amino acid sequence. The changes in the DNA sequence ofVP24 consisted of a transversion from G to C at nt 10795, a transversionfrom C to G at nt 10796, a transversion from T to A at nt 10846, atransversion from A to T at nt 10847, a transversion from C to G at nt11040, and a transversion from C to G at nt 11041. The changes in theamino acid sequence of VP24 consisted of a Cys to Ser change at position151, a Leu to His change at position 168, and a Pro to Gly change atposition 233 (SEQ ID NO: 19).

[0033] Two different sequences for the Ebola virus VP30 gene, VP30 andVP30#2 (SEQ ID NOS: 4 and 7) are included. Both of these sequencesdiffer from the GenBank sequence by the insertion of an A residue in theupstream noncoding sequence between nt 8469 and 8470 and an insertion ofa T residue between nt 9275 and 9276 that results in a change in theopen reading frame of VP30 and VP30#2 after position 255 (SEQ ID NOS: 20and 23). As a result, the C-terminus of the VP30 protein differssignificantly from that previously reported. In addition to these 2changes, the VP30#2 nucleic acid in SEQ ID NO:7 contains a conservativetransition from T to C at nt 9217. Because the primers originally usedto clone the VP30 gene into the replicon were designed based on theGenBank sequence, the first clone that we constructed (SEQ ID NO: 4) didnot contain what we believe to be the authentic C-terminus of theprotein. Therefore, in the absence of the VP30 stop codon, theC-terminal codon was replaced with 37 amino acids derived from thevector sequence. The resulting VP30 construct therefore differed fromthe GenBank sequence in that it contained 32 amino acids of VP30sequence (positions 256 to 287, SEQ ID NO:20) and 37 amino acids ofirrelevant sequence (positions 288 to 324, SEQ ID NO:20) in the place ofthe C-terminal 5 amino acids reported in GenBank. However, inclusion of37 amino acids of vector sequence in place of the C-terminal amino acid(Pro, SEQ ID NO: 23) did not inhibit the ability of the protein to serveas a protective antigen in BALB/c mice. We have also determined that aVEE replicon construct, which contains the authentic C-terminus of VP30(VP30#2, SEQ ID NO: 23), protects mice against a lethal Ebola challenge.

[0034] The nucleotide sequence for Ebola virus VP35 (SEQ ID NO:5)differed from the GenBank sequence by a transition from T to C at nt4006, a transition from T to C at nt 4025, and an insertion of a Tresidue between nt 4102 and 4103. These sequence changes resulted in achange from a Ser to a Pro at position 293 and a change from Phe to Serat position 299 (SEQ ID NO: 21). The insertion of the T residue resultedin a change in the open reading frame of VP35 from that previouslyreported by Sanchez et al. (1993) following amino acid number 324. As aresult, Ebola virus VP35 encodes a protein of 340 amino acids, whereamino acids 325 to 340 (SEQ ID NO: 21) differ from and replace theC-terminal 27 amino acids of the previously published sequence.

[0035] Sequencing of VP30 and VP35 was also performed on RT/PCR productsfrom RNA derived from cells that were infected with Ebola virus 1976,Ebola virus 1995 or the mouse-adapted Ebola virus. The changes notedabove for the Vrep constructs were also found in these Ebola viruses.Thus, we believe that these changes are real events and not artifacts ofcloning.

[0036] The Ebola virus VP40 nucleotide sequence (SEQ ID NO:6) differedfrom the GenBank sequence by a transversion from a C to G at nt 4451 anda transition from a G to A at nt 5081. These sequence changes did notalter the protein sequence of VP40 (SEQ ID NO: 22) from that of thepublished sequence.

[0037] Each of the Ebola virus genes were individually inserted into aVEE virus replicon vector. The VP24, VP30, VP35, and VP40 genes of EbolaZaire 1976 (Mayinga isolate) were cloned by reverse transcription of RNAfrom Ebola-infected Vero E6 cells and viral cDNAs were amplified usingthe polymerase chain reaction. The Ebola Zaire 1976 (Mayinga isolate) GPand NP genes were obtained from plasmids already containing these genes(Sanchez, A. et al., (1989) Virology 170, 81-91; Sanchez, A. etal.,(1993) Virus Res. 29, 215-240) and were subcloned into the VEEreplicon vector.

[0038] After characterization of the Ebola gene products expressed fromthe VEE replicon constructs in cell culture, these constructs werepackaged into infectious VEE virus replicon particles (VRPs) andsubcutaneously injected into BALB/c and C57BL/6 mice. As controls inthese experiments, mice were also immunized with a VEE repliconexpressing Lassa nucleoprotein (NP) as an irrelevant control antigen, orinjected with PBS buffer alone. The results of this study demonstratethat VRPs expressing the Ebola GP, NP, VP24, VP30, VP35 or VP40 genesinduced protection in mice and may reasonably to expected to provideprotection in humans.

[0039] DNA or polynucleotide sequences to which the invention alsorelates include sequences of at least about 6 nucleotides, preferably atleast about 8 nucleotides, more preferably at least about 10-12nucleotides, most preferably at least about 15-20 nucleotidescorresponding, i.e., homologous to or complementary to, a region of theEbola nucleotide sequences described above. Preferably, the sequence ofthe region from which the polynucleotide is derived is homologous to orcomplementary to a sequence which is unique to the Ebola genes. Whetheror not a sequence is unique to the Ebola gene can be determined bytechniques known to those of skill in the art. For example, the sequencecan be compared to sequences in databanks, e.g., GenBank and compared byDNA:DNA hybridization. Regions from which typical DNA sequences may bederived include but are not limited to, for example, regions encodingspecific epitopes, as well as non-transcribed and/or non-translatedregions.

[0040] The derived polynucleotide is not necessarily physically derivedfrom the nucleotide sequences shown in SEQ ID NO:1-7, but may begenerated in any manner, including for example, chemical synthesis orDNA replication or reverse transcription or transcription, which arebased on the information provided by the sequence of bases in theregion(s) from which the polynucleotide is derived. In addition,combinations of regions corresponding to that of the designated sequencemay be modified in ways known in the art to be consistent with anintended use. The sequences of the present invention can be used indiagnostic assays such as hybridization assays and polymerase chainreaction assays, for example, for the discovery of other Ebolasequences.

[0041] In another embodiment, the present invention relates to arecombinant DNA molecule that includes a vector and a DNA sequence asdescribed above. The vector can take the form of a plasmid, a eukaryoticexpression vector such as pcDNA3.1, pRcCMV2, pZeoSV2,or pCDM8, which areavailable from Invitrogen, or a virus vector such as baculovirusvectors, retrovirus vectors or adenovirus vectors, alphavirus vectors,and others known in the art.

[0042] In a further embodiment, the present invention relates to hostcells stably transformed or transfected with the above-describedrecombinant DNA constructs. The host cell can be prokaryotic (forexample, bacterial), lower eukaryotic (for example, yeast or insect) orhigher eukaryotic (for example, all mammals, including but not limitedto mouse and human). Both prokaryotic and eukaryotic host cells may beused for expression of the desired coding sequences when appropriatecontrol sequences which are compatible with the designated host areused.

[0043] Among prokaryotic hosts, E. coli is the most frequently used hostcell for expression. General control sequences for prokaryotes includepromoters and ribosome binding sites. Transfer vectors compatible withprokaryotic hosts are commonly derived from a plasmid containing genesconferring ampicillin and tetracycline resistance (for example, pBR322)or from the various pUC vectors, which also contain sequences conferringantibiotic resistance. These antibiotic resistance genes may be used toobtain successful transformants by selection on medium containing theappropriate antibiotics. Please see e.g., Maniatis, Fitsch and Sambrook,Molecular Cloning; A Laboratory Manual (1982) or DNA Cloning, Volumes Iand II (D. N. Glover ed. 1985) for general cloning methods. The DNAsequence can be present in the vector operably linked to sequencesencoding an IgG molecule, an adjuvant, a carrier, or an agent for aid inpurification of Ebola proteins, such as glutathione S-transferase.

[0044] In addition, the Ebola virus gene products can also be expressedin eukaryotic host cells such as yeast cells and mammalian cells.Saccharomyces cerevisiae, Saccharomyces carlsbergensis, and Pichiapastoris are the most commonly used yeast hosts. Control sequences foryeast vectors are known in the art. Mammalian cell lines available ashosts for expression of cloned genes are known in the art and includemany immortalized cell lines available from the American Type CultureCollection (ATCC), such as CHO cells, Vero cells, baby hamster kidney(BHK) cells and COS cells, to name a few. Suitable promoters are alsoknown in the art and include viral promoters such as that from SV40,Rous sarcoma virus (RSV), adenovirus (ADV), bovine papilloma virus(BPV), and cytomegalovirus (CMV). Mammalian cells may also requireterminator sequences, poly A addition sequences, enhancer sequenceswhich increase expression, or sequences which cause amplification of thegene. These sequences are known in the art.

[0045] The transformed or transfected host cells can be used as a sourceof DNA sequences described above. When the recombinant molecule takesthe form of an expression system, the transformed or transfected cellscan be used as a source of the protein described below.

[0046] In another embodiment, the present invention relates to Ebolavirion proteins such as

[0047] GP having an amino acid sequence corresponding to SEQ ID NO:17encompassing 676 amino acids,

[0048] NP, having an amino acid sequence corresponding to SEQ ID NO:18encompassing 739 amino acids,

[0049] VP24, having an amino acid sequence corresponding to SEQ ID NO:19encompassing 251 amino acids,

[0050] VP30, having an amino acid sequence corresponding SEQ ID NO:20encompassing 324 amino acids,

[0051] VP35, having an amino acid sequence corresponding to SEQ ID NO:21encompassing 340 amino acids, and

[0052] VP40, having an amino acid sequence corresponding to SEQ IDNO:22, encompassing 326 amino acids, and

[0053] VP30#2, having an amino acid sequence corresponding to SEQ IDNO:23 encompassing 288 amino acids, or any allelic variation of theseamino acid sequences. By allelic variation is meant a natural orsynthetic change in one or more amino acids which occurs betweendifferent serotypes or strains of Ebola virus and does not affect theantigenic properties of the protein. There are different strains ofEbola (Zaire 1976, Zaire 1995, Reston, Sudan, and Ivory Coast). The NPand VP genes of all these different viruses have not been sequenced. Itwould be expected that these proteins would have homology amongdifferent strains and that vaccination against one Ebola virus strainmight afford cross protection to other Ebola virus strains.

[0054] A polypeptide or amino acid sequence derived from any of theamino acid sequences in SEQ ID NO:17, 18, 19, 20, 21, 22, and 23 refersto a polypeptide having an amino acid sequence identical to that of apolypeptide encoded in the sequence, or a portion thereof wherein theportion consists of at least 2-5 amino acids, preferably at least 8-10amino acids, and more preferably at least 11-15 amino acids, or which isimmunologically identifiable with a polypeptide encoded in the sequence.

[0055] A recombinant or derived polypeptide is not necessarilytranslated from a designated nucleic acid sequence, or the DNA sequencefound in GenBank accession number L11365. It may be generated in anymanner, including for example, chemical synthesis, or expression from arecombinant expression system.

[0056] When the DNA or RNA sequences described above are in a repliconexpression system, such as the VEE replicon described above, theproteins can be expressed in vivo. The DNA sequence for any of the GP,NP, VP24, VP30, VP35, and VP40 virion proteins can be cloned into themultiple cloning site of a replicon such that transcription of the RNAfrom the replicon yields an infectious RNA encoding the Ebola protein orproteins of interest (see FIGS. 2A, 2B and 2C). The replicon constructsinclude Ebola virus GP (SEQ ID NO:1) cloned into a VEE replicon(VRepEboGP), Ebola virus NP (SEQ ID NO:2) cloned into a VEE replicon(VRepEboNP), Ebola virus VP24 (SEQ ID NO:3) cloned into a VEE replicon(VRepEboVP24), Ebola virus VP30 (SEQ ID NO:4) or VP30#2 (SEQ ID NO:7)cloned into a VEE replicon (VRepEboVP30 or VRepEboVP30(#2)), Ebola virusVP35 (SEQ ID NO:5) cloned into a VEE replicon (VRepEboVP35), and Ebolavirus VP40 (SEQ ID NO:6) cloned into a VEE replicon (VRepEboVP40). Thereplicon DNA or RNA can be used as a vaccine for inducing protectionagainst infection with Ebola.

[0057] Use of helper RNAs containing sequences necessary for packagingof the viral replicon transcripts will result in the production ofvirus-like particles containing replicon RNAs (FIG. 3). These packagedreplicons will infect host cells and initiate a single round ofreplication resulting in the expression of the Ebola proteins in theinfected cells. The packaged replicon constructs (i.e. VEE virusreplicon particles, VRP) include those that express Ebola virus GP(EboGPVRP), Ebola virus NP (EboNPVRP), Ebola virus VP24 (EboVP24VRP),Ebola virus VP30 (EboVP30VRP or EboVP30VRP(#2)), Ebola virus VP35(EboVP35VRP), and Ebola virus VP40 (EboVP40VRP).

[0058] In another embodiment, the present invention relates to RNAmolecules resulting from the transcription of the constructs describedabove. The RNA molecules can be prepared by in vitro transcription usingmethods known in the art and described in the Examples below.Alternatively, the RNA molecules can be produced by transcription of theconstructs in vivo, and isolating the RNA. These and other methods forobtaining RNA transcripts of the constructs are known in the art. Pleasesee Current Protocols in Molecular Biology. Frederick M. Ausubel et al.(eds.), John Wiley and Sons, Inc. The RNA molecules can be used, forexample, as a direct RNA vaccine, or to transfect cells along with RNAfrom helper plasmids, one of which expresses VEE glycoproteins and theother VEE capsid proteins, as described above, in order to obtainreplicon particles.

[0059] In a further embodiment, the present invention relates to amethod of producing the recombinant or fusion protein which includesculturing the above-described host cells under conditions such that theDNA fragment is expressed and the recombinant or fusion protein isproduced thereby. The recombinant or fusion protein can then be isolatedusing methodology well known in the art. The recombinant or fusionprotein can be used as a vaccine for immunity against infection withEbola or as a diagnostic tool for detection of Ebola infection.

[0060] In another embodiment, the present invention relates toantibodies specific for the above-described recombinant proteins (orpolypeptides). For instance, an antibody can be raised against a peptidehaving the amino acid sequence of any of SEQ ID NO:17-25, or against aportion thereof of at least 10 amino acids, preferably, 11-15 aminoacids. Persons with ordinary skill in the art using standard methodologycan raise monoclonal and polyclonal antibodies to the protein(orpolypeptide) of the present invention, or a unique portion thereof.Materials and methods for producing antibodies are well known in the art(see for example Goding, In Monoclonal Antibodies: Principles andPractice, Chapter 4, 1986).

[0061] In another embodiment, the present invention relates to an Ebolavaccine comprising VRPs that express one or more of the Ebola proteinsdescribed above. The vaccine is administered to a subject wherein thereplicon is able to initiate one round of replication producing theEbola proteins to which a protective immune response is initiated insaid subject.

[0062] It is likely that the protection afforded by these genes is dueto both the humoral (antibodies (Abs)) and cellular (cytotoxic T cells(CTLs)) arms of the immune system. Protective immunity induced to aspecific protein may comprise humoral immunity, cellular immunity, orboth. The only Ebola virus protein known to be on the outside of thevirion is the GP. The presence of GP on the virion surface makes it alikely target for GP-specific Abs that may bind either extracellularvirions or infected cells expressing GP on their surfaces. Serumtransfer studies in this invention demonstrate that Abs that recognizeGP protect mice against lethal Ebola virus challenge.

[0063] In contrast, transfer of Abs specific for NP, VP24, VP30, VP35,or VP40 did not protect mice against lethal Ebola challenge. This data,together with the fact that these are internal virion proteins that arenot readily accessible to Abs on either extracellular virions or thesurface of infected cells, suggest that the protection induced in miceby these proteins is mediated by CTLs.

[0064] CTLs can bind to and lyse virally infected cells. This processbegins when the proteins produced by cells are routinely digested intopeptides. Some of these peptides are bound by the class I or class IImolecules of the major histocompatability complex (MHC), which are thentransported to the cell surface. During virus infections, viral proteinsproduced within infected cells also undergo this process. CTLs that havereceptors that bind to both a specific peptide and the MHC moleculeholding the peptide lyse the peptide-bearing cell, thereby limitingvirus replication. Thus, CTLs are characterized as being specific for aparticular peptide and restricted to a class I or class II MHC molecule.

[0065] CTLs may be induced against any of the Ebola virus proteins, asall of the viral proteins are produced and digested within the infectedcell. Thus, protection to Ebola virus involves CTLs against GP, NP,VP24, VP30, VP35, and/or VP40. It is especially noteworthy that the VPproteins varied in their protective efficacy when tested in geneticallyinbred mice that differ at the MHC locus. This, together with theinability to demonstrate a role for Abs in protection induced by the VPproteins and the data in Table A below, demonstrates a role for CTLs.Thus, in this invention a vaccine may include several Ebola virusproteins (e.g., at least two), or several CTL epitopes (e.g., at leasttwo), capable of inducing broad protection to different Ebola viruses inoutbred populations (e.g. people). To that end, the inventors haveidentified 18 sequences recognized by CTLs, as determined initially bymeasuring gamma interferon production by intracellular cytokine stainingand gamma interferon secretion by the ELISpot assay. The ability to lysecells was measured in chromium release assays and protection wasevaluated by adoptive transfer of cells into Ebola-naïve mice. Wheresequence information is available, the conservation of these CTLepitopes in other Ebola viruses is noted in Table A. Conserved sequencesshould be capable of inducing protective CTLs to each of the viruses inwhich the sequence is present.

[0066] The identified CTL epitopes are:

[0067] Ebola virus NP SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:27, and SEQID NO:28;

[0068] Ebola virus GP SEQ ID NO:29 (encompassing YFGPAAEGI, SEQ IDNO:42);

[0069] Ebola virus VP24 SEQ ID NO:25 (encompassing KFINKLDAL, SEQ IDNO:43), SEQ ID NO:30 (encompassing NYNGLLSSI, SEQ ID NO:44), and SEQ IDNO:31 (encompassing PGPAKFSLL, SEQ ID NO:45);

[0070] Ebola virus VP30 SEQ ID NO:32 (encompassing LSLLCETHLR, SEQ IDNO:46), and SEQ ID NO:33 (encompassing MFITAFLNI, SEQ ID NO:47);

[0071] Ebola virus VP35 SEQ ID NO:34, SEQ ID NO:35, and SEQ ID NO:36;and

[0072] Ebola virus VP40 SEQ ID NO:37 (encompassing EFVLPPVQL, SEQ IDNO:48), SEQ ID NO:38 (encompassing FLVPPV, SEQ ID NO:49 and QYFTFDLTALK,SEQ ID NO:50), SEQ ID NO:39 (encompassing TSPEKIQAI, SEQ ID NO:51); SEQID NO:40 (encompassing RIGNQAFL, SEQ ID NO:52), and SEQ ID NO:41(encompassing QAFLQEFV, SEQ ID NO:53).

[0073] See Table A below.

[0074] Testing to identify the role of CTLs in protection was performedby obtaining CTLs from mice, expanding them in vitro, and transferringthe cells into an unvaccinated mouse of the same genetic background.Several hours later, the recipient mice were challenged with 10-1000 pfuof mouse-adapted Ebola virus. They were observed for signs of illnessfor 28 days. Control animals received no cells or cells that were notspecific for Ebola virus. Testing of the cells in vitro indicated thatthey were CD8+, a marker indicating class I-restriction. The inventorswere able to demonstrate that CTLs to these sequences protected at least80% of recipient mice from challenge. In all of our examples, theability to lyse peptide-pulsed target cells has predicted protection inmice receiving those cells. TABLE A Ebola Virus Epitopes Recognized byMurine CD8+T cells % INF-γ INF-γ Conserved Protein^(a) Epitope^(b)ICC^(c) ELISpot^(d) ⁵¹Cr^(e) Protective^(f) Restriction^(g) Strains^(h)GP WIPYFGPAAEGIYTE (SEQ ID 29) 0.40/0.08 Y Neg IP H-2^(b) R,G NPVYQVNNLEEIC (SEQ ID 24) 1.06/0.11 Y 55.6 Yes H-2^(b) G GQFLFASL (SEQ ID26) 0.88/0.11 Y 45   Yes H-2^(b) S,G DAVLYYHMM (SEQ ID 27) 0.99/0.11 Y40.6 Yes H-2^(b) G SFKAALSSL (SEQ ID 28) 0.63/0.04 Y 38.8 Yes H-2^(d)VP24 NILKFINKLDALHVV (SEQ ID 25) 0.52/0.09 Y 45.9 Yes H-2^(d) GNYNGLLSSIEGTQN (SEQ ID 30) 0.38/0.09 Y 50.6 Yes H-2^(d) R,GRMKPGPAKFSLLHESTLKAFTQGSS 3.34/0.09 Y 43.2 Yes* H-2^(d) R,G (SEQ ID 31)VP30 FSKSQLSLLCETHLR (SEQ ID 32) 0.45/0.15 N 47.3 Yes* H-2^(b)DLQSLIMFITAFLNI (SEQ ID 33) 0.7/0.15 N ND Yes* H-2^(b) VP35 RNIMYDHL(SEQ ID 34) 1.53/0.22 Y 87.4 Yes H-2^(b) MVAKYDLL (SEQ ID 35) 1.63/0.22N 78.9 Yes H-2^(b) R CDIENNPGL (SEQ ID 36) 1.99/0.15 N 80.4 Yes H-2^(b)VP40 AFLQEFVLPPVOLPQ (SEQ ID 37) 0.45/0.22 ND ND IP H-2^(d)FVLPPVQLPQYFTFDLTALK (SEQ ID 38) 0.41/0.22 ND 38   Yes* H-2^(d)KSGKKGNSADLTSPEKIOAIMTSLQDFKIV 0.6/0.22 N 36.4 IP H-2^(d) (SEQ ID 39)PLRLLRIGNQAFLQE (SEQ ID 40) 0.7/0.05 N 52.8 Yes H-2^(b) RIGNQAFLOEFVLPP(SEQ ID 41) 0.38/0.05 N 46.7 IP H-2^(b)

[0075] In another embodiment, the invention relates to a vaccine againstEbola infection including at least one of these CTL epitope sequences,and preferably at least one CTL epitope having the amino acid sequenceof SEQ ID NOs:24-53. Preferably, the vaccine includes at least two ofthe CTL epitope sequences, more preferably at least three, morepreferably at least four, more preferably at least five, and morepreferably all of the sequences. As shown in the examples below,protection is increased as the number of CTL epitopes in the immunogeniccomposition or vaccine is increased, and also as the number of epitopesfrom different Ebola proteins is increased.

[0076] In another embodiment, the vaccine includes a CTL epitopesequence from at least two different proteins selected from the groupconsisting of GP, NP, VP24, VP30, VP35 and VP40. More preferably, thevaccine includes a CTL epitope sequence from at least three differentproteins from that group, more preferably at least four, more preferablyat least five, and most preferably includes at least one CTL epitopesequence from each of the six proteins. The CTL epitopes may have theamino acid sequences as set forth in SEQ ID NOs:24-53. It is noted thatadministering the GP peptide alone may prevent the induction ofprotective antibodies, which may be undesirable.

[0077] In a further vaccine embodiment, the vaccine comprises virusreplicon particles (preferably VEE virus replicon particles but otheralphavirus replicon particles will do as described above) expressing theEbola virus GP, NP, VP24, VP30, VP35, or VP40 proteins, or anycombination of different VEE virus replicons each expressing one or moredifferent Ebola proteins selected from GP, NP, VP24, VP30, VP35 andVP40. For instance, in a preferred embodiment the Ebola VRPs express oneor more of the peptides specified by SEQ ID NOs: 24-53.

[0078] In another vaccine embodiment, the vaccine may include at aminimum at least one of the Ebola proteins selected from GP, NP, VP24,VP30, VP35 and VP40, but preferably contains at least two, morepreferably at least three, more preferably at least four, morepreferably at least five, and most preferably all of them. It is notedagain that administering the GP peptide alone may prevent the inductionof protective antibodies, which may be undesirable. For instance, in apreferred embodiment the vaccine includes at least the VP30, VP35 andVP40 proteins. In another preferred embodiment, the vaccine may includeone or more of SEQ ID NOs: 24-53.

[0079] When considering which type of vaccine may be most effective foran individual, it is noted that the same protective response could beinduced by the peptide or the full protein produced from the VRPs.Production of the peptide intracellularly is generally preferred becauseit is usually (but not always) more effective than providing itextracellularly. Thus, vaccines containing VRPs may be preferred becausethe VRPs infect cells and therefore achieve intracellular production.

[0080] Such vaccines might be delivered as synthetic peptides, or asfusion proteins, alone or co-administered with cytokines and/oradjuvants or carriers safe for human use, e.g. aluminum hydroxide, toincrease immunogenicity. In addition, sequences such as ubiquitin can beadded to increase antigen processing for more effective CTL responses.

[0081] In yet another embodiment, the present invention relates to amethod for providing immunity against Ebola virus, said methodcomprising administering one or more VRPs expressing any combination ofthe GP, NP, VP24, VP30 or VP30#2, VP35 and VP40 Ebola proteins to asubject such that a protective immune reaction is generated. In anotherrelated embodiment, the method may entail administering one or more VRPsexpressing any combination of the peptides designated SEQ ID NOs:24-53,or simply one or more of the peptides designated SEQ ID NOs:24-53.

[0082] Vaccine formulations of the present invention comprise animmunogenic amount of a VRP, such as for example EboVP24VRP describedabove, or, for a multivalent vaccine, a combination of replicons, in apharmaceutically acceptable carrier. An “immunogenic amount” is anamount of the VRP(s) sufficient to evoke an immune response in thesubject to which the vaccine is administered. An amount of from about10⁴-10⁸ focus-forming units per dose is suitable, depending upon the ageand species of the subject being treated. The subject may be inoculated2-3 times. Exemplary pharmaceutically acceptable carriers include, butare not limited to, sterile pyrogen-free water and sterile pyrogen-freephysiological saline solution.

[0083] Administration of the VRPs disclosed herein may be carried out byany suitable means, including parenteral injection (such asintraperitoneal, subcutaneous, or intramuscular injection), in ovoinjection of birds, orally, or by topical application of the virus(typically carried in a pharmaceutical formulation) to an airwaysurface. Topical application of the virus to an airway surface can becarried out by intranasal administration (e.g., by use of dropper, swab,or inhaler which deposits a pharmaceutical formulation intranasally).Topical application of the virus to an airway surface can also becarried out by inhalation administration, such as by creating respirableparticles of a pharmaceutical formulation (including both solidparticles and liquid particles) containing the replicon as an aerosolsuspension, and then causing the subject to inhale the respirableparticles. Methods and apparatus for administering respirable particlesof pharmaceutical formulations are well known, and any conventionaltechnique can be employed. Oral administration may be in the form of aningestable liquid or solid formulation.

[0084] When the replicon RNA or DNA is used as a vaccine, the repliconRNA or DNA can be administered directly using techniques such asdelivery on gold beads (gene gun), delivery by liposomes, or directinjection, among other methods known to people in the art. Any one ormore DNA constructs or replicating RNA described above can be use in anycombination effective to elicit an immunogenic response in a subject.Generally, the nucleic acid vaccine administered may be in an amount ofabout 1-5 ug of nucleic acid per dose and will depend on the subject tobe treated, capacity of the subject's immune system to develop thedesired immune response, and the degree of protection desired. Preciseamounts of the vaccine to be administered may depend on the judgement ofthe practitioner and may be peculiar to each subject and antigen.

[0085] The vaccine may be given in a single dose schedule, or preferablya multiple dose schedule in which a primary course of vaccination may bewith 1-10 separate doses, followed by other doses given at subsequenttime intervals required to maintain and or reinforce the immuneresponse, for example, at 1-4 months for a second dose, and if needed, asubsequent dose(s) after several months. Examples of suitableimmunization schedules include: (i) 0, 1 months and 6 months, (ii) 0, 7days and 1 month, (iii) 0 and 1 month, (iv) 0 and 6 months, (v) 0, 1 and2 months, or other schedules sufficient to elicit the desired immuneresponses expected to confer protective immunity, or reduce diseasesymptoms, or reduce severity of disease.

[0086] In a further embodiment, the present invention relates to amethod of detecting the presence of antibodies against Ebola virus in asample. Using standard methodology well known in the art, a diagnosticassay can be constructed by coating on a surface (i.e. a solid supportfor example, a microtitration plate, a membrane (e.g. nitrocellulosemembrane) or a dipstick), all or a unique portion of any of the Ebolaproteins described above or any combination thereof, and contacting itwith the serum of a person or animal suspected of having Ebola. Thepresence of a resulting complex formed between the Ebola protein(s) andserum antibodies specific therefor can be detected by any of the knownmethods common in the art, such as fluorescent antibody spectroscopy orcolorimetry. This method of detection can be used, for example, for thediagnosis of Ebola infection and for determining the degree to which anindividual has developed virus-specific antibodies after administrationof a vaccine.

[0087] In yet another embodiment, the present invention relates to amethod for detecting the presence of Ebola virion proteins in a sample.Antibodies against GP, NP, and the VP proteins could be used fordiagnostic assays. Using standard methodology well known in the art, adiagnostics assay can be constructed by coating on a surface (i.e. asolid support, for example, a microtitration plate or a membrane (e.g.nitrocellulose membrane)), antibodies specific for any of the Ebolaproteins described above, and contacting it with serum or a tissuesample of a person suspected of having Ebola infection. The presence ofa resulting complex formed between the protein or proteins in the serumand antibodies specific therefor can be detected by any of the knownmethods common in the art, such as fluorescent antibody spectroscopy orcolorimetry. This method of detection can be used, for example, for thediagnosis of Ebola virus infection.

[0088] In another embodiment, the present invention relates to adiagnostic kit which contains any combination of the Ebola proteinsdescribed above and ancillary reagents that are well known in the artand that are suitable for use in detecting the presence of antibodies toEbola in serum or a tissue sample. Tissue samples contemplated can befrom monkeys, humans, or other mammals.

[0089] In yet another embodiment, the present invention relates to DNAor nucleotide sequences for use in detecting the presence of Ebola virususing the reverse transcription-polymerase chain reaction (RT-PCR). TheDNA sequence of the present invention can be used to design primerswhich specifically bind to the viral RNA for the purpose of detectingthe presence of Ebola virus or for measuring the amount of Ebola virusin a sample. The primers can be any length ranging from 7 to 400nucleotides, preferably at least 10 to 15 nucleotides, or morepreferably 18 to 40 nucleotides. Reagents and controls necessary for PCRreactions are well known in the art. The amplified products can then beanalyzed for the presence of viral sequences, for example by gelfractionation, with or without hybridization, by radiochemistry, andimmunochemistry techniques.

[0090] In yet another embodiment, the present invention relates to adiagnostic kit which contains PCR primers specific for Ebola virus andancillary reagents for use in detecting the presence or absence of Ebolain a sample using PCR. Samples contemplated can be obtained from human,animal, e.g., horse, donkey, pig, mouse, hamster, monkey, or othermammals, birds, and insects, such as mosquitoes.

[0091] The following examples are included to demonstrate preferredembodiments of the invention. It should be appreciated by those of skillin the art that the techniques disclosed in the examples which followrepresent techniques discovered by the inventors and thought to functionwell in the practice of the invention, and thus can be considered toconstitute preferred modes for its practice. However, those of skill inthe art should, in light of the present disclosure, appreciate that manychanges can be made in the specific embodiments which are disclosed andstill obtain a like or similar result without departing from the spiritand scope of the invention.

[0092] The following MATERIALS AND METHODS were used in the examplesthat follow.

[0093] Cells Lines and Viruses

[0094] BHK (ATCC CCL 10), Vero 76 (ATCC CRL 1587), and Vero E6 (ATCC CRL1586) cell lines were maintained in minimal essential medium withEarle's salts, 5-10% fetal bovine serum, and 50 □g/mL gentamicinsulfate. For CTL assays, EL4 (ATCC TIB39), L5178Y (ATCC CRL 1723) andP815 (ATCC TIB64) were maintained in Dulbecco's minimal essential mediumsupplemented with 5-10% fetal bovine serum and antibiotics.

[0095] A stock of the Zaire strain of Ebola virus originally isolatedfrom a patient in the 1976 outbreak (Mayinga) and passagedintracerebrally 3 times in suckling mice and 2 times in Vero cells wasadapted to adult mice through serial passage in progressively oldersuckling mice (Bray et al.,(1998) J. Infect. Dis. 178, 651-661). Aplaque-purified ninth-mouse-passage isolate which was uniformly lethalfor adult mice (“mouse-adapted virus”) was propagated in Vero E6 cells,aliquotted, and used in all mouse challenge experiments andneutralization assays.

[0096] A stock of the Zaire strain of Ebola 1976 virus was passagedspleen to spleen in strain 13 guinea pigs four times. This guineapig-adapted strain was used to challenge guinea pigs.

[0097] Construction and Packaging of Recombinant VEE Virus Replicons(VRPs)

[0098] Replicon RNAs were packaged into VRPs as described (Pushko etal., 1997, supra). Briefly, capped replicon RNAs were produced in vitroby T7 run-off transcription of NotI-digested plasmid templates using theRiboMAX T7 RNA polymerase kit (Promega). BHK cells were co-transfectedwith the replicon RNAs and the 2 helper RNAs expressing the structuralproteins of the VEE virus. The cell culture supernatants were harvestedapproximately 30 hours after transfection and the replicon particleswere concentrated and purified by centrifugation through a 20% sucrosecushion. The pellets containing the packaged replicon particles weresuspended in PBS and the titers were determined by infecting Vero cellswith serial dilutions of the replicon particles and enumerating theinfected cells by indirect immunofluorescence with antibodies specificfor the Ebola proteins.

[0099] Immunoprecipitation of Ebola Virus Proteins Expressed from VEEVirus Replicons

[0100] BHK cells were transfected with either the Ebola virus GP, NP,VP24, VP30, VP35, or VP40 replicon RNAs. At 24 h post-transfection, theculture medium was replaced with minimal medium lacking cysteine andmethionine, and proteins were labeled for 1 h with ³⁵S-labeledmethionine and cysteine. Cell lysates or supernatants (supe) werecollected and immunoprecipitated with polyclonal rabbit anti-Ebola virusserum bound to protein A beads. ³⁵S-labeled Ebola virus structuralproteins from virions grown in Vero E6 cells were alsoimmunoprecipitated as a control for each of the virion proteins.Immunoprecipitated proteins were resolved by electrophoresis on an 11%SDS-polyacrylamide gel and were visualized by autoradiography.

[0101] Vaccination of Mice with VEE Virus Replicons

[0102] Groups of 10 BALB/c or C57BL/6 mice per experiment weresubcutaneously injected at the base of the neck with 2×10⁶ focus-formingunits of VRPs encoding the Ebola virus genes. As controls, mice werealso injected with either a control VRP encoding the Lassa nucleoprotein(NP) or with PBS. For booster inoculations, animals received identicalinjections at 1 month intervals. Data are recorded as the combinedresults of 2 or 3 separate experiments.

[0103] Ebola Infection of Mice

[0104] One month after the final booster inoculation, mice weretransferred to a BSL-4 containment area and challenged byintraperitoneal (ip) inoculation of 10 plaque-forming units (pfu) ofmouse-adapted Ebola virus (approximately 300 times the dose lethal for50% of adult mice). The mice were observed daily, and morbidity andmortality were recorded. Animals surviving at day 21 post-infection wereinjected again with the same dose of virus and observed for another 21days.

[0105] In some experiments, 4 or 5 mice from vaccinated and controlgroups were anesthetized and exsanguinated on day 4 (BALB/c mice) or day5 (C57BL/6 mice) following the initial viral challenge. The viral titersin individual sera were determined by plaque assay.

[0106] Passive Transfer of Immune Sera to Naive Mice.

[0107] Donor sera were obtained 28 days after the third inoculation with2×10⁶ focus-forming units of VRPs encoding the indicated Ebola virusgene, the control Lassa NP gene, or from unvaccinated control mice. OnemL of pooled donor sera was administered intraperitoneally (ip) tonaive, syngeneic mice 24 h prior to intraperitoneal challenge with 10pfu of mouse-adapted Ebola virus.

[0108] Vaccination and Challenge of Guinea Pigs.

[0109] EboGPVRP or EboNPVRP (1×10⁷ focus-forming units in 0.5 ml PBS)were administered subcutaneously to inbred strain 2 or strain 13 guineapigs (300-400 g). Groups of five guinea pigs were inoculated on days 0and 28 at one (strain 2) or two (strain 13) dorsal sites. Strain 13guinea pigs were also boosted on day 126. One group of Strain 13 guineapigs was vaccinated with both the GP and NP constructs. Blood sampleswere obtained after vaccination and after viral challenge. Guinea pigswere challenged on day 56 (strain 2) or day 160 (strain 13) bysubcutaneous administration of 1000 LD₅₀ (1×10⁴ PFU) of guineapig-adapted Ebola virus. Animals were observed daily for 60 days, andmorbidity (determined as changes in behavior, appearance, and weight)and survival were recorded. Blood samples were taken on the daysindicated after challenge and viremia levels were determined by plaqueassay.

[0110] Virus Titration and Neutralization Assay.

[0111] Viral stocks were serially diluted in growth medium, adsorbedonto confluent Vero E6 cells in 6- or 12-well dishes, incubated for 1hour at 37° C., and covered with an agarose overlay (Moe, J. et al.(1981) J. Clin. Microbiol. 13:791-793). A second overlay containing 5%neutral red solution in PBS or agarose was added 6 days later, andplaques were counted the following day. Pooled pre-challenge serumsamples from some of the immunized groups were tested for the presenceof Ebola-neutralizing antibodies by plaque reduction neutralizationassay. Aliquots of Ebola virus in growth medium were mixed with serialdilutions of test serum, or with normal serum, or medium only, incubatedat 37° C. for 1 h, and used to infect Vero E6 cells. Plaques werecounted 1 week later.

[0112] Cytotoxic T Cell Assays.

[0113] BALB/c and C57BL/6 mice were inoculated with VRPs encoding Ebolavirus NP or VP24 or the control Lassa NP protein. Mice were euthanizedat various times after the last inoculation and their spleens removed.The spleens were gently ruptured to generate single cell suspensions.Spleen cells (1×10⁶/ml) were cultured in vitro for 2 days in thepresence of 10-25 □M of peptides synthesized from Ebola virus NP or VP24amino acid sequences, and then for an additional 5 days in the presenceof peptide and 10% supernatant from concanavalin A-stimulated syngeneicspleen cells. Synthetic peptides were made from Ebola virus amino acidsequences predicted by a computer algorithm (HLA Peptide BindingPredictions, Parker, K. C., et al. (1994) J. Immunol. 152:163) to have alikelihood of meeting the MHC class I binding requirements of the BALB/c(H-2^(d)) and C57BL/6 (H-2^(b)) haplotypes. Only 2 of 8 peptidespredicted by the algorithm and tested to date have been identified ascontaining CTL epitopes. After in vitro restimulation, the spleen cellswere tested in a standard ⁵¹chromium-release assay well known in the art(see, for example, Hart et al. (1991) Proc. Natl. Acad. Sci. USA 88:9449-9452). Percent specific lysis of peptide-coated, MHC-matched ormismatched target cells was calculated as:$\frac{{{Experimental}\quad {cpm}} - {{Spontaneous}\quad {cpm} \times 100}}{{{Maximum}\quad {cpm}} - {{Spontaneous}\quad {cpm}}}$

[0114] Spontaneous cpm are the number of counts released from targetcells incubated in medium. Maximum cpm are obtained by lysing targetcells with 1% Triton X-100. Experimental cpm are the counts from wellsin which target cells are incubated with varying numbers of effector(CTL) cells. Target cells tested were L5178Y lymphoma or P815mastocytoma cells (MHC matched to the H2^(d) BALB/c mice and EL4lymphoma cells (MHC matched to the H2^(b) C57BL/6 mice). Theeffector:target (E:T) ratios tested were 25:1, 12:1, 6:1 and 3:1.

EXAMPLE 1 Survival of Mice Inoculated with VRPs Encoding Ebola Proteins

[0115] Mice were inoculated two or three times at 1 month intervals with2×10⁶ focus-forming units of VRPs encoding individual Ebola virus genes,or Lassa virus NP as a control, or with phosphate buffered saline (PBS).Mice were challenged with 10 pfu of mouse-adapted Ebola virus one monthafter the final immunization. The mice were observed daily, andmorbidity and mortality data are shown in Table 1A for BALB/c mice andTable 1B for C57BL/6 mice. The viral titers in individual sera of somemice on day 4 (BALB/c mice) or day 5 (C57BL/6 mice) following theinitial viral challenge were determined by plaque assay. TABLE 1Survival Of Mice Inoculated With VRPs Encoding Ebola Proteins VRP#Injections S/T¹ (%) MDD² V/T³ Viremia⁴ A. BALB/c Mice EboNP 330/30(100%) 5/5 5.2 2 19/20(95%) 7 5/5 4.6 EboGP 3 15/29(52%) 8 1/5 6.62 14/20(70%) 7 3/5 3.1 EboVP24 3 27/30(90%) 8 5/5 5.2 2 19/20(95%) 6 4/44.8 EboVP30 3 17/20(85%) 7 5/5 6.2 2 11/20(55%) 7 5/5 6.5 EboVP35 35/19(26%) 7 5/5 6.9 2 4/20(20%) 7 5/5 6.5 EboVP40 3 14/20(70%) 8 5/5 4.62 17/20(85%) 7 5/5 5.6 LassaNP 3 0/29(0%) 7 5/5 8.0 2 0/20(0%) 7 5/5 8.4none(PBS) 3 1/30(3%) 6 5/5 8.3 2 0/20(0%) 6 5/5 8.7 B. C57BL/6 MiceEboNP 3 15/20(75%) 8 5/5 4.1 2 8/10(80%) 9 ND⁵ ND EboGP 3 19/20(95%) 100/5 — 2 10/10(100%) — ND ND EboVP24 3 0/20(0%) 7 5/5 8.6 EboVP30 32/20(10%) 8 5/5 7.7 EboVP35 3 14/20(70%) 8 5/5 4.5 EboVP40 3 1/20(5%) 74/4 7.8 LassaNP 3 1/20(5%) 7 4/4 8.6 2 0/10(0%) 7 ND ND none(PBS) 33/20(15%) 7 5/5 8.6 2 0/10(0%) 7 ND ND

EXAMPLE 2 VP24-Immunized BALB/c Mice Survive a High-Dose Challenge withEbola Virus

[0116] BALB/c mice were inoculated two times with 2×10⁶ focus-formingunits of EboVP24VRP. Mice were challenged with either 1×10³ pfu or 1×10⁵pfu of mouse-adapted Ebola virus 1 month after the second inoculation.Morbidity and mortality data for these mice are shown in Table 2. TABLE2 VP24-Immunized BALB/c Mice Survive A High-Dose Challenge With Ebolavirus Replicon Challenge Dose Survivors/Total EboVP24 1 × 10³ pfu 5/5 (3× 10⁴ LD₅₀) EboVP24 1 × 10⁵ pfu 5/5 (3 × 10⁶ LD₅₀) None 1 × 10³ pfu 0/4(3 × 10⁴ LD₅₀) None 1 × 10⁵ pfu 0/3 (3 × 10⁶ LD₅₀)

EXAMPLE 3 Passive Transfer of Immune Sera Can Protect Naive Mice from aLethal Challenge of Ebola Virus

[0117] Donor sera were obtained 28 days after the third inoculation with2×10⁶ focus-forming units of VRPs encoding the indicated Ebola virusgene, the control Lassa NP gene, or from unvaccinated control mice. OnemL of pooled donor sera was administered intraperitoneally (ip) tonaive, syngeneic mice 24 h prior to intraperitoneal challenge with 10pfu of mouse-adapted Ebola virus. TABLE 3 Passive Transfer of ImmuneSera Can Protect Unvaccinated Mice from a Lethal Challenge of EbolaVirus Specificity of Survivors/ Mean Day Donor Sera Total of Death A.BALB/c Mice Ebola GP 15/20  8 Ebola NP 1/20 7 Ebola VP24 0/20 6 EbolaVP30 0/20 7 Ebola VP35 ND¹ ND Ebola VP40 0/20 6 LassaNP 0/20 7 Normalmouse sera 0/20 6 B. C57BL/6 Mice Ebola GP 17/20  7 Ebola NP 0/20 7Ebola VP24 ND ND Ebola VP30 ND ND Ebola VP35 0/20 7 Ebola VP40 ND NDLassaNP 0/20 7 Normal mouse sera O/20 7

EXAMPLE 4 Immunogenicity and Efficacy of VRepEboGP and VRepEboNP inGuinea Pigs

[0118] EboGPVRP or EboNPVRP (1×10⁷ IU in 0.5 ml PBS) were administeredsubcutaneously to inbred strain 2 or strain 13 guinea pigs (300-400 g).Groups of five guinea pigs were inoculated on days 0 and 28 at one(strain 2) or two (strain 13) dorsal sites. Strain 13 guinea pigs werealso boosted on day 126. One group of Strain 13 guinea pigs wasvaccinated with both the GP and NP constructs. Blood samples wereobtained after vaccination and after viral challenge.

[0119] Sera from vaccinated animals were assayed for antibodies to Ebolaby plaque-reduction neutralization, and ELISA. Vaccination withVRepEboGP or NP induced high titers of antibodies to the Ebola proteins(Table 4) in both guinea pig strains. Neutralizing antibody responseswere only detected in animals vaccinated with the GP construct (Table4).

[0120] Guinea pigs were challenged on day 56 (strain 2) or day 160(strain 13) by subcutaneous administration of 1000 LD₅₀ (10⁴ PFU) ofguinea pig-adapted Ebola virus. Animals were observed daily for 60 days,and morbidity (determined as changes in behavior, appearance, andweight) and survival were recorded. Blood samples were taken on the daysindicated after challenge and viremia levels were determined by plaqueassay. Strain 13 guinea pigs vaccinated with the GP construct, alone orin combination with NP, survived lethal Ebola challenge (Table 4).Likewise, vaccination of strain 2 inbred guinea pigs with the GPconstruct protected 3/5 animals against death from lethal Ebolachallenge, and significantly prolonged the mean day of death (MDD) inone of the two animals that died (Table 4). Vaccination with NP alonedid not protect either guinea pig strain. TABLE 4 Immunogenicity andefficacy of VRepEboGP and VRepEboNP in guinea pigs Survivors/Viremia^(c) VRP ELISA^(a) PRNT₅₀ total(MDD^(b)) d7 d14 A. Strain 2guinea pigs GP 4.1 30 3/5(13 + 2.8) 2.3 1.8 NP 3.9 <10 0/5(9.2 + 1.1)3.0 — Mock <1.5 <10 0/5(8.8 + 0.5) 3.9 — B. Strain 13 guinea pigs GP 4.0140 5/5 <2.0 <2.0 GP/NP 3.8 70 5/5 <2.0 <2.0 NP 2.8 <10 1/5(8.3 + 2.2)4.6 — LassaNP <1.5 <10 2/5(8.3 + 0.6) 4.8 —

EXAMPLE 5 Induction of Murine CTL Responses to Ebola Virus NP and EbolaVirus VP24 Proteins

[0121] BALB/c and C57BL/6 mice were inoculated with VRPs encoding Ebolavirus NP or VP24. Mice were euthanized at various times after the lastinoculation and their spleens removed. Spleen cells (1×10⁶/ml) werecultured in vitro for 2 days in the presence of 10 to 25 □M of peptides,and then for an additional 5 days in the presence of peptide and 10%supernatant from concanavalin A-stimulated syngeneic spleen cells. Afterin vitro restimulation, the spleen cells were tested in a standard⁵¹chromium-release assay. Percent specific lysis of peptide-coated,MHC-matched or mismatched target cells was calculated as:$\frac{{{Experimental}\quad {cpm}} - {{Spontaneous}\quad {cpm} \times 100}}{{{Maximum}\quad {cpm}} - {{Spontaneous}\quad {cpm}}}$

[0122] In the experiments shown, spontaneous release did not exceed 15%.TABLE 5 Induction of murine CTL responses to Ebola virus NP and Ebolavirus VP24 proteins. % Specific Lysis E:T ratio Mice, VRP¹ Peptide²Cell³ 25 BALB/c, VP24 None P815 55 BALB/c, VP24 SEQ ID NO: 25 P815 93C57BL/6, EboNP None EL4 2 C57BL/6, EboNP⁴ SEQ ID NO: 24 EL4 70 C57BL/6,EboNP LassaNP EL4 2 C57BL/6, LassaNP None L5178Y 1 C57BL/6, LassaNP SEQID NO: 24 L5178Y 0 C57BL/6, LassaNP None EL4 2 C57BL/6, LassaNP SEQ IDNO: 24 EL4 6

EXAMPLE 6 Induction of Murine T Cell Responses that Protect AgainstEbola Challenge

[0123] Mice and injections. BALB/c and C57Bl/6 mice were injected scwith 2×10⁶ IU of VEE virus replicons encoding either the individualEbola genes or Lassa NP (3 injections 1 month apart). The genes used tomake replicons are from the human Zaire76 virus. One month after thefinal immunization, mice were transferred to BSL-4 containment andchallenged by ip inoculation of 10 or 1000 pfu (300 or 30000 LD₅₀) ofmouse-adapted Ebola Zaire. This virus has amino acid changes in NP at nt683 (S to G), VP35 at nt 3163 (A to V), VP24 at nt 10493 (T to I), andin L at nt 14380 (F to L) and nt 16174 (I to V). There are three othernt changes, including an insertion in the intergenic region at nt 10343.GenBank accession number AF499101.

[0124] T cell assays. Single cell suspensions were prepared from spleensby passage through cell 70 μM strainers. Spleen cells were depleted oferythrocytes by treatment with buffered ammonium chloride solution andenumerated by trypan blue exclusion on a hemacytometer. For in vitrorestimulations, 1-5 μg peptide(s) and human recombinant IL-2 (10 U/ml,National Cancer Institute) were added to a cell density of 1×10⁶/ml andthe cultures incubated 4-7 days. For intracellular IFN-γ staining,splenocytes were cultured at 37° C. for 5 hr with 1-5 μg of peptide(s)or PMA (25 ng/ml) and ionomycin (1.25 ug/ml) in 100 μl complete mediumcontaining 10 μg/ml brefeldin A (BFA). After culture, the cells wereblocked with mAbs to FcRIII/II receptor and stained with αCD44 FITC andanti-CD8 Cychrome (Pharmingen, San Diego, Calif.) in PBS/BFA. The cellswere then fixed in 1% formaldehyde (Ted Pella, Redding, Calif.),permeabilized with PBS containing 0.5% saponin, and stained with αIFN-γPE (Pharmingen, San Diego, Calif.). The data were acquired using aFACSCalibur flow cytometer and analyzed with CELLQuest software(Becton-Dickinson. Cytotoxicity assays were performed using target cells(EL4, L5178Y) labeled with ⁵¹Cr (Na₂CrO₄; New England Nuclear, Boston,Mass.) and pulsed with peptide for 1.5 hours. Unpulsed target cells wereused as negative controls. Various numbers of effector cells wereincubated with 2500 target cells for 4 hours. Percentage specificrelease was calculated as: % specificrelease=(experimental−spontaneous)/(maximum−spontaneous)×100.Spontaneous release values were obtained by incubation of target cellsin medium alone and were routinely <10% of maximum release. Maximumrelease values were obtained by the addition of 100 μl 1% TritonX-100.

[0125] Adoptive transfer experiments. After in vitro restimulation,cells are Ficoll purified, washed three times with 0.3Mmethyl-a-D-mannopyranoside and twice with complete media. Cells arecounted and adjusted to 25.0×10{circumflex over ( )}6 cells/ml inendotoxin-free PBS. A total volume of 0.2 mls is given to each mouse byi.p. injection 4 hr before challenge with 1000 PFU of mouse-adaptedEbola virus. Animals are observed and sickness or death is noted ondaily charts.

[0126] As shown in Table A, this data identifies the protectivemechanism induced by VRP vaccination, showing the role of T cells. Itindicates the ability to predict protection from in vitro assays,specifically the intracellular cytokine and chromium release assays.Thus, a positive ICC result is reasonably predictive of conferredprotection, even if the protection is listed as incomplete; the rest ofthe data strongly indicate protection. Notably, where the CTL sequencesare conserved between Zaire and the other Ebola viruses,cross-protection may reasonably be inferred. (The protection in Table Arefers to adoptive transfer of CTLs to unvaccinated mice beforechallenge, not the vaccination with a certain protein.)

EXAMPLE 7 Determination of Interference in Protection by MultipleReplicons

[0127] The purpose of this experiment was to determine if multiple VEEreplicons that do not provide complete protection (VP24, 30, 40 in BL/6mice) will interfere with a protective replicon Ebola NP that hasdefined CTL epitopes. Co-administration did not interfere with theinduction of protection. Vaccine Survival VRep EBOV NP, VP24, VP30, VP4010/10 VRep EBOV NP 6/6 PBS 0/7

[0128] The CTLs to NP are CD8(+),and recognize epitopes SEQ ID NO:24, 26and 27. Evaluation of the ability to lyse peptide-pulsed target cellswas assessed using spleen cells from mice vaccinated with the EBOCVRepNP alone, or all four replicons (NP, VP24, VP30 and VP40). Althoughresponses were somewhat lower in the mice receiving four replicons, thethreshold of immunity was maintained. % Lysis of target cells coatedwith peptide (background on untreated target cells is subtracted)Vaccine Effector/target ratio NP-1 NP-8 NP-17 NP 100:1  55 31 64 50:1 6123 45 25:1 67 15 31 12:1 51 13 21 Mix 100:1  41 40 45 50:1 37 32 28 25:126 33 19 12:1 19 18 12

EXAMPLE 8 Improved Efficacy Induced by a Cocktail Formulation ofSuboptimal EBOV Vrep

[0129] In studies where we examined the protective efficacy of repliconsindividually, we observed that some replicons (such as VP30, VP24 andVP40) protected fewer than 100% of the mice. When protection was lessthan 50%, we suggested that the protein was not particularly protectivefor that mouse strain. However, in some cases, we did observe that20-30% of the mice survived, suggesting that we might be able tooptimize our vaccine strategy to provide protection with those proteins.As we are evaluating a cocktail formulation, we approached this issue byinjecting mice with combinations of the three VP replicons than had poorefficacy in C57Bl/6 mice.

[0130] C57BL/6 mice were injected SC at the base of the neck with2.0×10⁶ packaged VEE virus replicon particles for each Ebola VP protein,then rested for 27 days and then boosted twice at days 28 and 56. On day84, mice were injected intraperitoneally with approximately 3×10⁴ LD50(1000 PFU) of mouse-adapted Ebola virus. Strain Vaccine Survival C57BL/6VRep VP35 30/30 C57BL/6 VReps VP35, VP30, VP24, VP40 30/30 C57BL/6Medium  0/23 C57BL/6 VReps VP30, VP24, VP40 28/30

[0131] The data indicate that combining the three VP replicons providedsignificantly better protection than when we administered them singly.Of note, the VP24 replicon has never protected a single C57BL/6 mousewhen administered alone and is not likely contributing to protection.However, importantly, its inclusion in the formulation also does notinterfere with induction of protective responses to the other VPs.

[0132] These data shows that a cocktail formulation may be a preferredvaccine because it induces a broader array of T cells (i.e. CTLs tomultiple proteins) and that, together, these may meet the thresholdneeded for protection. We expect the same phenomenon will apply tonon-human primate studies. This also provides support for the inclusionof multiple Ebola peptides/proteins in a cocktail formulation. As anexample, if a human infected with Ebola needs 1 million effectors, butvaccination induces only 400,000 to each protein, it may be additive tohave 1.2 million spread across 3 proteins. Otherwise, waiting one dayfor that person's cells to divide to 800,000 and a second day to cross 1million—but that would likely be too late for survival.

[0133] Ebola Zaire 1976 (Mayinga) virus causes acute hemorrhagic fevercharacterized by high mortality. There are no current vaccines oreffective therapeutic measures to protect individuals who are exposed tothis virus. In addition, it is not known which genes are essential forevoking protective immunity and should therefore be included in avaccine designed for human use. In this study, the GP, NP, VP24, VP30,VP35, and VP40 virion protein genes of the Ebola Zaire 1976 (Mayinga)virus were cloned and inserted into a Venezuelan equine encephalitis(VEE) virus replicon vector (VRep) as shown in FIGS. 2A and 2B. TheseVReps were packaged as VEE replicon particles (VRPs) using the VEE virusstructural proteins provided as helper RNAs, as shown in FIG. 3. Thisenables expression of the Ebola virus proteins in host cells. The Ebolavirus proteins produced from these constructs were characterized invitro and were shown to react with polyclonal rabbit anti-Ebola virusantibodies bound to Protein A beads following SDS gel electrophoresis ofimmunoprecipitated proteins (FIG. 4).

[0134] The Ebola virus genes were sequenced from the VEE replicon clonesand are listed here as SEQ ID NO:1 (GP), 2 (NP), 3 (VP24), 4 (VP30), 5(VP35), 6 (VP40), and 7 (VP30#2) as described below. The correspondingamino acid sequences of the Ebola proteins expressed from thesereplicons are listed as SEQ ID NO: 17, 18, 19, 20, 21, 22, and 23,respectively. Changes in the DNA sequence relative to the sequencepublished by Sanchez et al. (1993) are described relative to thenucleotide (nt) sequence number from GenBank (accession number L11365).

[0135] The sequence we obtained for Ebola virus GP (SEQ ID NO:1)differed from the GenBank sequence by a transition from A to G at nt8023. This resulted in a change in the amino acid sequence from Ile toVal at position 662 (SEQ ID NO: 17).

[0136] The DNA sequence we obtained for Ebola virus NP (SEQ ID NO:2)differed from the GenBank sequence at the following 4 positions:insertion of a C residue between nt 973 and 974, deletion of a G residueat nt 979, transition from C to T at nt 1307, and a transversion from Ato C at nt 2745. These changes resulted in a change in the proteinsequence from Arg to Glu at position 170 and a change from Leu to Phe atposition 280 (SEQ ID NO: 18).

[0137] The Ebola virus VP24 (SEQ ID NO:3) gene differed from the GenBanksequence at 6 positions, resulting in 3 nonconservative changes in theamino acid sequence. The changes in the DNA sequence of VP24 consistedof a transversion from G to C at nt 10795, a transversion from C to G atnt 10796, a transversion from T to A at nt 10846, a transversion from Ato T at nt 10847, a transversion from C to G at nt 11040, and atransversion from C to G at nt 11041. The changes in the amino acidsequence of VP24 consisted of a Cys to Ser change at position 151, a Leuto His change at position 168, and a Pro to Gly change at position 233(SEQ ID NO: 19).

[0138] We have included 2 different sequences for the Ebola virus VP30gene (SEQ ID NOS:4 and SEQ ID NO:7). Both of these sequences differ fromthe GenBank sequence by the insertion of an A residue in the upstreamnoncoding sequence between nt 8469 and 8470 and an insertion of a Tresidue between nt 9275 and 9276 that results in a change in the openreading frame of VP30 and VP30#2 after position 255 (SEQ ID NOS:20 andSEQ ID NO:23). As a result, the C-terminus of the VP30 protein differssignificantly from that previously reported. In addition to these 2changes, the VP30#2 gene in SEQ ID NO:23 contains a conservativetransition from T to C at nt 9217. Because the primers originally usedto clone the VP30 gene into the replicon were designed based on theGenBank sequence, the first clone that we constructed (SEQ ID NO:4) didnot contain what we believe to be the authentic C-terminus of theprotein. Therefore, in the absence of the VP30 stop codon, theC-terminal codon was replaced with 37 amino acids derived from thevector sequence. The resulting VP30 construct therefore differed fromthe GenBank sequence in that it contained 32 amino acids of VP30sequence (positions 256 to 287, SEQ ID NO:20) and 37 amino acids ofirrelevant sequence (positions 288 to 324, SEQ ID NO:20) in the place ofthe C-terminal 5 amino acids reported in GenBank. However, inclusion of37 amino acids of vector sequence in place of the C-terminal amino acid(Pro, SEQ ID NO:23) did not inhibit the ability of the protein to serveas a protective antigen in BALB/c mice. We have also determined that aVEE replicon construct (SEQ ID NO:7), which contains the authenticC-terminus of VP30 (VP30#2, SEQ ID NO:23), is able to protect miceagainst a lethal Ebola challenge.

[0139] The DNA sequence for Ebola virus VP35 (SEQ ID NO:5) differed fromthe GenBank sequence by a transition from T to C at nt 4006, atransition from T to C at nt 4025, and an insertion of a T residuebetween nt 4102 and 4103. These sequence changes resulted in a changefrom a Ser to a Pro at position 293 and a change from Phe to Ser atposition 299 (SEQ ID NO:21). The insertion of the T residue resulted ina change in the open reading frame of VP35 from that previously reportedby Sanchez et al. (1993) following amino acid number 324. As a result,Ebola virus VP35 encodes for a protein of 340 amino acids, where aminoacids 325 to 340 (SEQ ID NO:21) differ from and replace the C-terminal27 amino acids of the previously published sequence.

[0140] Sequencing of VP30 and VP35 was also performed on RT/PCR productsfrom RNA derived from cells that were infected with Ebola virus 1976,Ebola virus 1995 or the mouse-adapted Ebola virus. The changes notedabove for the VRep constructs were also found in these Ebola viruses.Thus, we believe that these changes are real events and not artifacts ofcloning.

[0141] The Ebola virus VP40 differed from the GenBank sequence by atransversion from a C to G at nt 4451 and a transition from a G to A atnt 5081. These sequence changes did not alter the protein sequence ofVP40 (SEQ ID NO:22) from that of the published sequence.

[0142] To evaluate the protective efficacy of individual Ebola virusproteins and to determine whether the major histocompatibility (MHC)genes influence the immune response to Ebola virus antigens, twoMHC-incompatible strains of mice were vaccinated with VRPs expressing anEbola protein. As controls for these experiments, some mice wereinjected with VRPs expressing the nucleoprotein of Lassa virus or wereinjected with phosphate-buffered saline (PBS). Following Ebola viruschallenge, the mice were monitored for morbidity and mortality, and theresults are shown in Table 1.

[0143] The GP, NP, VP24, VP30, and VP40 proteins of Ebola virusgenerated either full or partial protection in BALB/c mice, and maytherefore be useful components of a vaccine for humans or other mammals.Vaccination with VRPs encoding the NP protein afforded the bestprotection. In this case, 100% of the mice were protected after threeinoculations and 95% of the mice were protected after two inoculations.The VRP encoding VP24 also protected 90% to 95% of BALB/c mice againstEbola virus challenge. In separate experiments (Table 2), two or threeinoculations with VRPs encoding the VP24 protein protected BALB/c micefrom a high dose (1×10⁵ plaque—forming units (3×10⁶ LD50)) ofmouse-adapted Ebola virus.

[0144] Example 1 shows that vaccination with VRPs encoding GP protected52-70% of BALB/c mice. The lack of protection was not due to a failureto respond to the VRP encoding GP, as all mice had detectable Ebolavirus-specific serum antibodies after vaccination. Improved results werelater seen, which are thought to be dose-dependent. Further, as shown inExamples 6-8, combining suboptimal formulations gives dramaticallybetter protection.

[0145] Also in Example 1, some protective efficacy was further observedin BALB/c mice vaccinated two or three times with VRPs expressing theVP30 protein (55% and 85%, respectively),or the VP40 protein (70% and80%, respectively). The VP35 protein was not efficacious in the BALB/cmouse model, as only 20% and 26% of the mice were protected after eithertwo or three doses, respectively. Again, improved results were laterseen, which are thought to be dose-dependent; and we found thatcombining suboptimal formulations gives dramatically better protection(e.g., combination of VP24, VP30 and VP40).

[0146] Geometric mean titers of viremia were markedly reduced in BALB/cmice vaccinated with VRPs encoding Ebola virus proteins after challengewith Ebola virus, indicating an ability of the induced immune responsesto reduce virus replication (Table 1A). In this study, immune responsesto the GP protein were able to clear the virus to undetectable levelswithin 4 days after challenge in some mice.

[0147] When the same replicons were examined for their ability toprotect C57BL/6 mice from a lethal challenge of Ebola virus, only theGP, NP, and VP35 proteins were efficacious (Table 1B). The bestprotection, 95% to 100%, was observed in C57BL/6 mice inoculated withVRPs encoding the GP protein. Vaccination with VRPs expressing NPprotected 75% to 80% of the mice from lethal disease. In contrast towhat was observed in the BALB/c mice, the VP35 protein was the only VPprotein able to significantly protect the C57BL/6 mice. In this case, 3inoculations with VRPs encoding VP35 protected 70% of the mice fromEbola virus challenge. The reason behind the differences in protectionin the two mouse strains is believed to be due to the ability of theimmunogens to sufficiently stimulate the cellular immune system. As withthe BALB/c mice, the effects of the induced immune responses were alsoobserved in reduced viremias and, occasionally, in a prolonged time todeath of C57BL/6 mice.

[0148] Example 4 shows that VRPs expressing Ebola virus GP or NP werealso evaluated for protective efficacy in a guinea pig model. Sera fromvaccinated animals were assayed for antibodies to Ebola by westernblotting, IFA, plaque-reduction neutralization, and ELISA. Vaccinationwith either VRP (GP or NP) induced high titers of antibodies to theEbola proteins (Table 4) in both guinea pig strains. We later found thatVP40 induced high titers (4 logs) in mice. Neutralizing antibodyresponses were only detected in animals vaccinated with the VRPexpressing GP (Table 4).

[0149] As shown in Example 4, vaccination of strain 2 inbred guinea pigswith the GP construct protected 3/5 animals against death from lethalEbola challenge, and significantly prolonged the mean day of death inone of the two animals that died (Table 4). All of the strain 13 guineapigs vaccinated with the GP construct, alone or in combination with NP,survived lethal Ebola challenge (Table 4). Vaccination with NP alone didnot protect either guinea pig strain from challenge with the guineapig-adapted Ebola virus. Of note, guinea pigs are also inbred, and thefailure of NP to protect may indicate that they could not respond withappropriate T cells, but could make protective antibodies to GP. This isfurther support for our preferred embodiments including multiplepeptides and proteins, and even all six of the Ebola proteins.

[0150] As shown in Example 3, to identify the immune mechanisms thatmediate protection against Ebola virus and to determine whetherantibodies are sufficient to protect against lethal disease, passivetransfer studies were performed. One mL of immune sera, obtained frommice previously vaccinated with one of the Ebola virus VRPs, waspassively administered to unvaccinated mice 24 hours before challengewith a lethal dose of mouse-adapted Ebola virus. Antibodies to GP, butnot to NP or the VP proteins, protected mice from an Ebola viruschallenge (Table 3). Antibodies to GP protected 75% of the BALB/c miceand 85% of the C57BL/6 mice from death. When the donor sera wereexamined for their ability to neutralize Ebola virus in aplaque-reduction neutralization assay, a 1:20 to 1:40 dilution of theGP-specific antisera reduced the number of viral plaque-forming units byat least 50% (data not shown). In contrast, antisera to the NP and VPproteins did not neutralize Ebola virus at a 1:20 or 1:40 dilution.These results are consistent with the finding that GP is the only viralprotein found on the surface of Ebola virus, and is likely to inducevirus-neutralizing antibodies.

[0151] As shown in Examples 5 and 6, cince the NP and VP proteins ofEbola virus are internal virion proteins to which antibodies are notsufficient for protection, it is likely that cytotoxic T lymphocytes(CTLs) are also important for protection against Ebola virus. Theinventors investigated cellular immune responses to individual Ebolavirus proteins expressed from VRPs identified CTL responses to the VP24and NP proteins (Table 5). One CTL epitope that we identified for theEbola virus NP is recognized by C57BL/6 (H-2^(b)) mice, and has an aminoacid sequence of, or contained within, the following 11 amino acids:VYQVNNLEEIC (SEQ ID NO:24). Vaccination with EboNPVRP and in vitrorestimulation of spleen cells with this peptide consistently inducesstrong CTL responses in C57BL/6 (H-2^(b)) mice. In vivo vaccination toEbola virus NP is required to detect the CTL activity, as evidenced bythe failure of cells from C57BL/6 mice vaccinated with Lassa NP todevelop lytic activity to peptide (SEQ ID NO:24) after in vitrorestimulation with it. Specific lysis has been observed using very loweffector:target ratios (<2:1). This CTL epitope is H-2^(b) restricted inthat it is not recognized by BALB/c (H-2^(d)) cells treated the same way(data not shown), and H-2^(b) effector cells will not lyseMHC-mismatched target cells coated with this peptide.

[0152] A CTL epitope in the VP24 protein was also identified. It isrecognized by BALB/c (H-2^(d)) mice, and has an amino acid sequence of,or contained within, the following 23 amino acids:LKFINKLDALLVVNYNGLLSSIF (SEQ ID NO:25). In the data shown in Table 5,high (>90%) specific lysis of P815 target cells coated with this peptidewas observed. The background lysis of cells that were not peptide-coatedwas also high (>50%), which is probably due to the activity of naturalkiller cells. We are planning to repeat this experiment using the L5178Ytarget cells, which are not susceptible to natural killer cells. Thisshows that CTLs mediated protection, which is further demonstrated bythe evidence in Examples 6, 7 and 8.

1 53 1 2298 DNA Ebola zaire 1 atcgataagc tcggaattcg agctcgcccggggatcctct agagtcgaca acaacacaat 60 gggcgttaca ggaatattgc agttacctcgtgatcgattc aagaggacat cattctttct 120 ttgggtaatt atccttttcc aaagaacattttccatccca cttggagtca tccacaatag 180 cacattacag gttagtgatg tcgacaaactagtttgtcgt gacaaactgt catccacaaa 240 tcaattgaga tcagttggac tgaatctcgaagggaatgga gtggcaactg acgtgccatc 300 tgcaactaaa agatggggct tcaggtccggtgtcccacca aaggtggtca attatgaagc 360 tggtgaatgg gctgaaaact gctacaatcttgaaatcaaa aaacctgacg ggagtgagtg 420 tctaccagca gcgccagacg ggattcggggcttcccccgg tgccggtatg tgcacaaagt 480 atcaggaacg ggaccgtgtg ccggagactttgccttccat aaagagggtg ctttcttcct 540 gtatgatcga cttgcttcca cagttatctaccgaggaacg actttcgctg aaggtgtcgt 600 tgcatttctg atactgcccc aagctaagaaggacttcttc agctcacacc ccttgagaga 660 gccggtcaat gcaacggagg acccgtctagtggctactat tctaccacaa ttagatatca 720 ggctaccggt tttggaacca atgagacagagtacttgttc gaggttgaca atttgaccta 780 cgtccaactt gaatcaagat tcacaccacagtttctgctc cagctgaatg agacaatata 840 tacaagtggg aaaaggagca ataccacgggaaaactaatt tggaaggtca accccgaaat 900 tgatacaaca atcggggagt gggccttctgggaaactaaa aaaaacctca ctagaaaaat 960 tcgcagtgaa gagttgtctt tcacagttgtatcaaacgga gccaaaaaca tcagtggtca 1020 gagtccggcg cgaacttctt ccgacccagggaccaacaca acaactgaag accacaaaat 1080 catggcttca gaaaattcct ctgcaatggttcaagtgcac agtcaaggaa gggaagctgc 1140 agtgtcgcat ctaacaaccc ttgccacaatctccacgagt ccccaatccc tcacaaccaa 1200 accaggtccg gacaacagca cccataatacacccgtgtat aaacttgaca tctctgaggc 1260 aactcaagtt gaacaagatc accgcagaacagacaacgac agcacagcct ccgacactcc 1320 ctctgccacg accgcagccg gacccccaaaagcagagaac accaacacga gcaagagcac 1380 tgacttcctg gaccccgcca ccacaacaagtccccaaaac cacagcgaga ccgctggcaa 1440 caacaacact catcaccaag ataccggagaagagagtgcc agcagcggga agctaggctt 1500 aattaccaat actattgctg gagtcgcaggactgatcaca ggcgggagaa gaactcgaag 1560 agaagcaatt gtcaatgctc aacccaaatgcaaccctaat ttacattact ggactactca 1620 ggatgaaggt gctgcaatcg gactggcctggataccatat ttcgggccag cagccgaggg 1680 aatttacata gaggggctaa tgcacaatcaagatggttta atctgtgggt tgagacagct 1740 ggccaacgag acgactcaag ctcttcaactgttcctgaga gccacaactg agctacgcac 1800 cttttcaatc ctcaaccgta aggcaattgatttcttgctg cagcgatggg gcggcacatg 1860 ccacattctg ggaccggact gctgtatcgaaccacatgat tggaccaaga acataacaga 1920 caaaattgat cagattattc atgattttgttgataaaacc cttccggacc agggggacaa 1980 tgacaattgg tggacaggat ggagacaatggataccggca ggtattggag ttacaggcgt 2040 tgtaattgca gttatcgctt tattctgtatatgcaaattt gtcttttagt ttttcttcag 2100 attgcttcat ggaaaagctc agcctcaaatcaatgaaacc aggatttaat tatatggatt 2160 acttgaatct aagattactt gacaaatgataatataatac actggagctt taaacatagc 2220 caatgtgatt ctaactcctt taaactcacagttaatcata aacaaggttt gagtcgacct 2280 gcagccaagc ttatcgat 2298 2 2428DNA Ebola zaire 2 atcgataagc ttggctgcag gtcgactcta gaggatccga gtatggattctcgtcctcag 60 aaaatctgga tggcgccgag tctcactgaa tctgacatgg attaccacaagatcttgaca 120 gcaggtctgt ccgttcaaca ggggattgtt cggcaaagag tcatcccagtgtatcaagta 180 aacaatcttg aagaaatttg ccaacttatc atacaggcct ttgaagcaggtgttgatttt 240 caagagagtg cggacagttt ccttctgatg ctttgtcttc atcatgcgtaccagggagat 300 tacaaacttt tcttggaaag tggcgcagtc aagtatttgg aagggcacgggttccgtttt 360 gaagtcaaga agcgtgatgg agtgaagcga cttgaggaat tgctgccagcagtatctagt 420 ggaaaaaaca ttaagagaac acttgctgcc atgccggaag aggagacaactgaagctaat 480 gccggtcagt ttctctcctt tgcaagtcta ttccttccga aattggtagtaggagaaaag 540 gcttgccttg agaaggttca aaggcaaatt caagtacatg cagagcaaggactgatacaa 600 tatccaacag cttggcaatc agtaggacac atgatggtga ttttccgtttgatgcgaaca 660 aattttctga tcaaatttct cctaatacac caagggatgc acatggttgccgggcatgat 720 gccaacgatg ctgtgatttc aaattcagtg gctcaagctc gtttttcaggcttattgatt 780 gtcaaaacag tacttgatca tatcctacaa aagacagaac gaggagttcgtctccatcct 840 cttgcaagga ccgccaaggt aaaaaatgag gtgaactcct ttaaggctgcactcagctcc 900 ctggccaagc atggagagta tgctcctttc gcccgacttt tgaacctttctggagtaaat 960 aatcttgagc atggtctttt ccctcaacta tcggcaattg cactcggagtcgccacagca 1020 cacgggagta ccctcgcagg agtaaatgtt ggagaacagt atcaacaactcagagaggct 1080 gccactgagg ctgagaagca actccaacaa tatgcagagt ctcgcgaacttgaccatctt 1140 ggacttgatg atcaggaaaa gaaaattctt atgaacttcc atcagaaaaagaacgaaatc 1200 agcttccagc aaacaaacgc tatggtaact ctaagaaaag agcgcctggccaagctgaca 1260 gaagctatca ctgctgcgtc actgcccaaa acaagtggac attacgatgatgatgacgac 1320 attccctttc caggacccat caatgatgac gacaatccta gccatcaagatgatgatccg 1380 actgactcac aggatacgac cattcccgat gtggtggttg atcccgatgatggaagctac 1440 ggcgaatacc agagttactc ggaaaacggc atgaatgcac cagatgacttggtcctattc 1500 gatctagacg aggacgacga ggacactaag ccagtgccta atagatcgaccaagggtgga 1560 caacagaaga acagtcaaaa gggccagcat atagagggca gacagacacaatccaggcca 1620 attcaaaatg tcccaggccc tcacagaaca atccaccacg ccagtgcgccactcacggac 1680 aatgacagaa gaaatgaacc ctccggctca accagccctc gcatgctgacaccaattaac 1740 gaagaggcag acccactgga cgatgccgac gacgagacgt ctagccttccgcccttggag 1800 tcagatgatg aagagcagga cagggacgga acttccaacc gcacacccactgtcgcccca 1860 ccggctcccg tatacagaga tcactctgaa aagaaagaac tcccgcaagacgagcaacaa 1920 gatcaggacc acactcaaga ggccaggaac caggacagtg acaacacccagtcagaacac 1980 tcttttgagg agatgtatcg ccacattcta agatcacagg ggccatttgatgctgttttg 2040 tattatcata tgatgaagga tgagcctgta gttttcagta ccagtgatggcaaagagtac 2100 acgtatccag actcccttga agaggaatat ccaccatggc tcactgaaaaagaggctatg 2160 aatgaagaga atagatttgt tacattggat ggtcaacaat tttattggccggtgatgaat 2220 cacaagaata aattcatggc aatcctgcaa catcatcagt gaatgagcatggaacaatgg 2280 gatgattcaa ccgacaaata gctaacatta agtagtccag gaacgaaaacaggaagaatt 2340 tttgatgtct aaggtgtgaa ttattatcac aataaaagtg attcttatttttgaatttgg 2400 gcgagctcga attcccgagc ttatcgat 2428 3 847 DNA Ebolazaire 3 atcgatctcc agacaccaag caagacctga gaaaaaacca tggctaaagctacgggacga 60 tacaatctaa tatcgcccaa aaaggacctg gagaaagggg ttgtcttaagcgacctctgt 120 aacttcttag ttagccaaac tattcagggg tggaaggttt attgggctggtattgagttt 180 gatgtgactc acaaaggaat ggccctattg catagactga aaactaatgactttgcccct 240 gcatggtcaa tgacaaggaa tctctttcct catttatttc aaaatccgaattccacaatt 300 gaatcaccgc tgtgggcatt gagagtcatc cttgcagcag ggatacaggaccagctgatt 360 gaccagtctt tgattgaacc cttagcagga gcccttggtc tgatctctgattggctgcta 420 acaaccaaca ctaaccattt caacatgcga acacaacgtg tcaaggaacaattgagccta 480 aaaatgctgt cgttgattcg atccaatatt ctcaagttta ttaacaaattggatgctcta 540 catgtcgtga actacaacgg attgttgagc agtattgaaa ttggaactcaaaatcataca 600 atcatcataa ctcgaactaa catgggtttt ctggtggagc tccaagaacccgacaaatcg 660 gcaatgaacc gcatgaagcc tgggccggcg aaattttccc tccttcatgagtccacactg 720 aaagcattta cacaaggatc ctcgacacga atgcaaagtt tgattcttgaatttaatagc 780 tctcttgcta tctaactaag gtagaatact tcatattgag ctaactcatatatgctgact 840 catcgat 847 4 973 DNA Ebola zaire 4 atcgatcaga tctgcgaaccggtagagttt agttgcaacc taacacacat aaagcattgg 60 tcaaaaagtc aatagaaatttaaacagtga gtggagacaa cttttaaatg gaagcttcat 120 atgagagagg acgcccacgagctgccagac agcattcaag ggatggacac gaccaccatg 180 ttcgagcacg atcatcatccagagagaatt atcgaggtga gtaccgtcaa tcaaggagcg 240 cctcacaagt gcgcgttcctactgtatttc ataagaagag agttgaacca ttaacagttc 300 ctccagcacc taaagacatatgtccgacct tgaaaaaagg atttttgtgt gacagtagtt 360 tttgcaaaaa agatcaccagttggagagtt taactgatag ggaattactc ctactaatcg 420 cccgtaagac ttgtggatcagtagaacaac aattaaatat aactgcaccc aaggactcgc 480 gcttagcaaa tccaacggctgatgatttcc agcaagagga aggtccaaaa attaccttgt 540 tgacactgat caagacggcagaacactggg cgagacaaga catcagaacc atagaggatt 600 caaaattaag agcattgttgactctatgtg ctgtgatgac gaggaaattc tcaaaatccc 660 agctgagtct tttatgtgagacacacctaa ggcgcgaggg gcttgggcaa gatcaggcag 720 aacccgttct cgaagtatatcaacgattac acagtgataa aggaggcagt tttgaagctg 780 cactatggca acaatgggacctacaatccc taattatgtt tatcactgca ttcttgaata 840 ttgctctcca gttaccgtgtgaaagttctg ctgtcgttgt ttcagggtta agaacattgg 900 ttcctcaatc agataatgaggaagcttcaa ccaacccggg gacatgctca tggtctgatg 960 agggtacatc gat 973 51148 DNA Ebola zaire 5 atcgatagaa aagctggtct aacaagatga caactagaacaaagggcagg ggccatactg 60 cggccacgac tcaaaacgac agaatgccag gccctgagctttcgggctgg atctctgagc 120 agctaatgac cggaagaatt cctgtaagcg acatcttctgtgatattgag aacaatccag 180 gattatgcta cgcatcccaa atgcaacaaa cgaagccaaacccgaagacg cgcaacagtc 240 aaacccaaac ggacccaatt tgcaatcata gttttgaggaggtagtacaa acattggctt 300 cattggctac tgttgtgcaa caacaaacca tcgcatcagaatcattagaa caacgcatta 360 cgagtcttga gaatggtcta aagccagttt atgatatggcaaaaacaatc tcctcattga 420 acagggtttg tgctgagatg gttgcaaaat atgatcttctggtgatgaca accggtcggg 480 caacagcaac cgctgcggca actgaggctt attgggccgaacatggtcaa ccaccacctg 540 gaccatcact ttatgaagaa agtgcgattc ggggtaagattgaatctaga gatgagaccg 600 tccctcaaag tgttagggag gcattcaaca atctaaacagtaccacttca ctaactgagg 660 aaaattttgg gaaacctgac atttcggcaa aggatttgagaaacattatg tatgatcact 720 tgcctggttt tggaactgct ttccaccaat tagtacaagtgatttgtaaa ttgggaaaag 780 atagcaactc attggacatc attcatgctg agttccaggccagcctggct gaaggagact 840 ctcctcaatg tgccctaatt caaattacaa aaagagttccaatcttccaa gatgctgctc 900 cacctgtcat ccacatccgc tctcgaggtg acattccccgagcttgccag aaaagcttgc 960 gtccagtccc accatcgccc aagattgatc gaggttgggtatgtgttttt cagcttcaag 1020 atggtaaaac acttggactc aaaatttgag ccaatctcccttccctccga aagaggcgaa 1080 taatagcaga ggcttcaact gctgaactat agggtacgttacattaatga tacacttgtg 1140 agatcgat 1148 6 1123 DNA Ebola zaire 6atcgatccta cctcggctga gagagtgttt tttcattaac cttcatcttg taaacgttga 60gcaaaattgt taaaaatatg aggcgggtta tattgcctac tgctcctcct gaatatatgg 120aggccatata ccctgtcagg tcaaattcaa caattgctag aggtggcaac agcaatacag 180gcttcctgac accggagtca gtcaatgggg acactccatc gaatccactc aggccaattg 240ccgatgacac catcgaccat gccagccaca caccaggcag tgtgtcatca gcattcatcc 300ttgaagctat ggtgaatgtc atatcgggcc ccaaagtgct aatgaagcaa attccaattt 360ggcttcctct aggtgtcgct gatcaaaaga cctacagctt tgactcaact acggccgcca 420tcatgcttgc ttcatacact atcacccatt tcggcaaggc aaccaatcca cttgtcagag 480tcaatcggct gggtcctgga atcccggatc atcccctcag gctcctgcga attggaaacc 540aggctttcct ccaggagttc gttcttccgc cagtccaact accccagtat ttcacctttg 600atttgacagc actcaaactg atcacccaac cactgcctgc tgcaacatgg accgatgaca 660ctccaacagg atcaaatgga gcgttgcgtc caggaatttc atttcatcca aaacttcgcc 720ccattctttt acccaacaaa agtgggaaga aggggaacag tgccgatcta acatctccgg 780agaaaatcca agcaataatg acttcactcc aggactttaa gatcgttcca attgatccaa 840ccaaaaatat catgggaatc gaagtgccag aaactctggt ccacaagctg accggtaaga 900aggtgacttc taaaaatgga caaccaatca tccctgttct tttgccaaag tacattgggt 960tggacccggt ggctccagga gacctcacca tggtaatcac acaggattgt gacacgtgtc 1020attctcctgc aagtcttcca gctgtgattg agaagtaatt gcaataattg actcagatcc 1080agttttatag aatcttctca gggatagtgc ataacatatc gat 1123 7 1165 DNA Ebolazaire 7 atcgatcaga tctgcgaacc ggtagagttt agttgcaacc taacacacataaagcattgg 60 tcaaaaagtc aatagaaatt taaacagtga gtggagacaa cttttaaatggaagcttcat 120 atgagagagg acgcccacga gctgccagac agcattcaag ggatggacacgaccaccatg 180 ttcgagcacg atcatcatcc agagagaatt atcgaggtga gtaccgtcaatcaaggagcg 240 cctcacaagt gcgcgttcct actgtatttc ataagaagag agttgaaccattaacagttc 300 ctccagcacc taaagacata tgtccgacct tgaaaaaagg atttttgtgtgacagtagtt 360 tttgcaaaaa agatcaccag ttggagagtt taactgatag ggaattactcctactaatcg 420 cccgtaagac ttgtggatca gtagaacaac aattaaatat aactgcacccaaggactcgc 480 gcttagcaaa tccaacggct gatgatttcc agcaagagga aggtccaaaaattaccttgt 540 tgacactgat caagacggca gaacactggg cgagacaaga catcagaaccatagaggatt 600 caaaattaag agcattgttg actctatgtg ctgtgatgac gaggaaattctcaaaatccc 660 agctgagtct tttatgtgag acacacctaa ggcgcgaggg gcttgggcaagatcaggcag 720 aacccgttct cgaagtatat caacgattac acagtgataa aggaggcagttttgaagctg 780 cactatggca acaatgggac cgacaatccc taatcatgtt tatcactgcattcttgaata 840 ttgctctcca gttaccgtgt gaaagttctg ctgtcgttgt ttcagggttaagaacattgg 900 ttcctcaatc agataatgag gaagcttcaa ccaacccggg gacatgctcatggtctgatg 960 agggtacccc ttaataaggc tgactaaaac actatataac cttctacttgatcacaatac 1020 tccgtatacc tatcatcata tatttaatca agacgatatc ctttaaaacttattcagtac 1080 tataatcact ctcgtttcaa attaataaga tgtgcatgat tgccctaatatatgaagagg 1140 tatgatacaa ccctaacaga tcgat 1165 8 30 DNA ArtificialSequence Description of Artificial Sequence Forward primer for VP24 8gggatcgatc tccagacacc aagcaagacc 30 9 33 DNA Artificial SequenceDescription of Artificial Sequence Reverse primer for VP24 9 gggatcgatgagtcagcata tatgagttag ctc 33 10 30 DNA Artificial Sequence Descriptionof Artificial Sequence Forward primer for VP30 10 cccatcgatc agatctgcgaaccggtagag 30 11 31 DNA Artificial Sequence Description of ArtificialSequence Reverse primer for VP30 11 cccatcgatg taccctcatc agaccatgag c31 12 33 DNA Artificial Sequence Description of Artificial SequenceForward primer for VP35 12 gggatcgata gaaaagctgg tctaacaaga tga 33 13 36DNA Artificial Sequence Description of Artificial Sequence Reverseprimer for VP35 13 cccatcgatc tcacaagtgt atcattaatg taacgt 36 14 30 DNAArtificial Sequence Description of Artificial Sequence Forward primerfor VP40 14 cccatcgatc ctacctcggc tgagagagtg 30 15 33 DNA ArtificialSequence Description of Artificial Sequence Reverse primer for VP40 15cccatcgata tgttatgcac tatccctgag aag 33 16 30 DNA Artificial SequenceDescription of Artificial Sequence Reverse primer for VP30#2 16cccatcgatc tgttagggtt gtatcatacc 30 17 676 PRT Ebola zaire 17 Met GlyVal Thr Gly Ile Leu Gln Leu Pro Arg Asp Arg Phe Lys Arg 1 5 10 15 ThrSer Phe Phe Leu Trp Val Ile Ile Leu Phe Gln Arg Thr Phe Ser 20 25 30 IlePro Leu Gly Val Ile His Asn Ser Thr Leu Gln Val Ser Asp Val 35 40 45 AspLys Leu Val Cys Arg Asp Lys Leu Ser Ser Thr Asn Gln Leu Arg 50 55 60 SerVal Gly Leu Asn Leu Glu Gly Asn Gly Val Ala Thr Asp Val Pro 65 70 75 80Ser Ala Thr Lys Arg Trp Gly Phe Arg Ser Gly Val Pro Pro Lys Val 85 90 95Val Asn Tyr Glu Ala Gly Glu Trp Ala Glu Asn Cys Tyr Asn Leu Glu 100 105110 Ile Lys Lys Pro Asp Gly Ser Glu Cys Leu Pro Ala Ala Pro Asp Gly 115120 125 Ile Arg Gly Phe Pro Arg Cys Arg Tyr Val His Lys Val Ser Gly Thr130 135 140 Gly Pro Cys Ala Gly Asp Phe Ala Phe His Lys Glu Gly Ala PhePhe 145 150 155 160 Leu Tyr Asp Arg Leu Ala Ser Thr Val Ile Tyr Arg GlyThr Thr Phe 165 170 175 Ala Glu Gly Val Val Ala Phe Leu Ile Leu Pro GlnAla Lys Lys Asp 180 185 190 Phe Phe Ser Ser His Pro Leu Arg Glu Pro ValAsn Ala Thr Glu Asp 195 200 205 Pro Ser Ser Gly Tyr Tyr Ser Thr Thr IleArg Tyr Gln Ala Thr Gly 210 215 220 Phe Gly Thr Asn Glu Thr Glu Tyr LeuPhe Glu Val Asp Asn Leu Thr 225 230 235 240 Tyr Val Gln Leu Glu Ser ArgPhe Thr Pro Gln Phe Leu Leu Gln Leu 245 250 255 Asn Glu Thr Ile Tyr ThrSer Gly Lys Arg Ser Asn Thr Thr Gly Lys 260 265 270 Leu Ile Trp Lys ValAsn Pro Glu Ile Asp Thr Thr Ile Gly Glu Trp 275 280 285 Ala Phe Trp GluThr Lys Lys Asn Leu Thr Arg Lys Ile Arg Ser Glu 290 295 300 Glu Leu SerPhe Thr Val Val Ser Asn Gly Ala Lys Asn Ile Ser Gly 305 310 315 320 GlnSer Pro Ala Arg Thr Ser Ser Asp Pro Gly Thr Asn Thr Thr Thr 325 330 335Glu Asp His Lys Ile Met Ala Ser Glu Asn Ser Ser Ala Met Val Gln 340 345350 Val His Ser Gln Gly Arg Glu Ala Ala Val Ser His Leu Thr Thr Leu 355360 365 Ala Thr Ile Ser Thr Ser Pro Gln Ser Leu Thr Thr Lys Pro Gly Pro370 375 380 Asp Asn Ser Thr His Asn Thr Pro Val Tyr Lys Leu Asp Ile SerGlu 385 390 395 400 Ala Thr Gln Val Glu Gln His His Arg Arg Thr Asp AsnAsp Ser Thr 405 410 415 Ala Ser Asp Thr Pro Ser Ala Thr Thr Ala Ala GlyPro Pro Lys Ala 420 425 430 Glu Asn Thr Asn Thr Ser Lys Ser Thr Asp PheLeu Asp Pro Ala Thr 435 440 445 Thr Thr Ser Pro Gln Asn His Ser Glu ThrAla Gly Asn Asn Asn Thr 450 455 460 His His Gln Asp Thr Gly Glu Glu SerAla Ser Ser Gly Lys Leu Gly 465 470 475 480 Leu Ile Thr Asn Thr Ile AlaGly Val Ala Gly Leu Ile Thr Gly Gly 485 490 495 Arg Arg Thr Arg Arg GluAla Ile Val Asn Ala Gln Pro Lys Cys Asn 500 505 510 Pro Asn Leu His TyrTrp Thr Thr Gln Asp Glu Gly Ala Ala Ile Gly 515 520 525 Leu Ala Trp IlePro Tyr Phe Gly Pro Ala Ala Glu Gly Ile Tyr Ile 530 535 540 Glu Gly LeuMet His Asn Gln Asp Gly Leu Ile Cys Gly Leu Arg Gln 545 550 555 560 LeuAla Asn Glu Thr Thr Gln Ala Leu Gln Leu Phe Leu Arg Ala Thr 565 570 575Thr Glu Leu Arg Thr Phe Ser Ile Leu Asn Arg Lys Ala Ile Asp Phe 580 585590 Leu Leu Gln Arg Trp Gly Gly Thr Cys His Ile Leu Gly Pro Asp Cys 595600 605 Cys Ile Glu Pro His Asp Trp Thr Lys Asn Ile Thr Asp Lys Ile Asp610 615 620 Gln Ile Ile His Asp Phe Val Asp Lys Thr Leu Pro Asp Gln GlyAsp 625 630 635 640 Asn Asp Asn Trp Trp Thr Gly Trp Arg Gln Trp Ile ProAla Gly Ile 645 650 655 Gly Val Thr Gly Val Val Ile Ala Val Ile Ala LeuPhe Cys Ile Cys 660 665 670 Lys Phe Val Phe 675 18 739 PRT Ebola zaire18 Met Asp Ser Arg Pro Gln Lys Ile Trp Met Ala Pro Ser Leu Thr Glu 1 510 15 Ser Asp Met Asp Tyr His Lys Ile Leu Thr Ala Gly Leu Ser Val Gln 2025 30 Gln Gly Ile Val Arg Gln Arg Val Ile Pro Val Tyr Gln Val Asn Asn 3540 45 Leu Glu Glu Ile Cys Gln Leu Ile Ile Gln Ala Phe Glu Ala Gly Val 5055 60 Asp Phe Gln Glu Ser Ala Asp Ser Phe Leu Leu Met Leu Cys Leu His 6570 75 80 His Ala Tyr Gln Gly Asp Tyr Lys Leu Phe Leu Glu Ser Gly Ala Val85 90 95 Lys Tyr Leu Glu Gly His Gly Phe Arg Phe Glu Val Lys Lys Arg Asp100 105 110 Gly Val Lys Arg Leu Glu Glu Leu Leu Pro Ala Val Ser Ser GlyLys 115 120 125 Asn Ile Lys Arg Thr Leu Ala Ala Met Pro Glu Glu Glu ThrThr Glu 130 135 140 Ala Asn Ala Gly Gln Phe Leu Ser Phe Ala Ser Leu PheLeu Pro Lys 145 150 155 160 Leu Val Val Gly Glu Lys Ala Cys Leu Glu LysVal Gln Arg Gln Ile 165 170 175 Gln Val His Ala Glu Gln Gly Leu Ile GlnTyr Pro Thr Ala Trp Gln 180 185 190 Ser Val Gly His Met Met Val Ile PheArg Leu Met Arg Thr Asn Phe 195 200 205 Leu Ile Lys Phe Leu Leu Ile HisGln Gly Met His Met Val Ala Gly 210 215 220 His Asp Ala Asn Asp Ala ValIle Ser Asn Ser Val Ala Gln Ala Arg 225 230 235 240 Phe Ser Gly Leu LeuIle Val Lys Thr Val Leu Asp His Ile Leu Gln 245 250 255 Lys Thr Glu ArgGly Val Arg Leu His Pro Leu Ala Arg Thr Ala Lys 260 265 270 Val Lys AsnGlu Val Asn Ser Phe Lys Ala Ala Leu Ser Ser Leu Ala 275 280 285 Lys HisGly Glu Tyr Ala Pro Phe Ala Arg Leu Leu Asn Leu Ser Gly 290 295 300 ValAsn Asn Leu Glu His Gly Leu Phe Pro Gln Leu Ser Ala Ile Ala 305 310 315320 Leu Gly Val Ala Thr Ala His Gly Ser Thr Leu Ala Gly Val Asn Val 325330 335 Gly Glu Gln Tyr Gln Gln Leu Arg Glu Ala Ala Thr Glu Ala Glu Lys340 345 350 Gln Leu Gln Gln Tyr Ala Glu Ser Arg Glu Leu Asp His Leu GlyLeu 355 360 365 Asp Asp Gln Glu Lys Lys Ile Leu Met Asn Phe His Gln LysLys Asn 370 375 380 Glu Ile Ser Phe Gln Gln Thr Asn Ala Met Val Thr LeuArg Lys Glu 385 390 395 400 Arg Leu Ala Lys Leu Thr Glu Ala Ile Thr AlaAla Ser Leu Pro Lys 405 410 415 Thr Ser Gly His Tyr Asp Asp Asp Asp AspIle Pro Phe Pro Gly Pro 420 425 430 Ile Asn Asp Asp Asp Asn Pro Gly HisGln Asp Asp Asp Pro Thr Asp 435 440 445 Ser Gln Asp Thr Thr Ile Pro AspVal Val Val Asp Pro Asp Asp Gly 450 455 460 Ser Tyr Gly Glu Tyr Gln SerTyr Ser Glu Asn Gly Met Asn Ala Pro 465 470 475 480 Asp Asp Leu Val LeuPhe Asp Leu Asp Glu Asp Asp Glu Asp Thr Lys 485 490 495 Pro Val Pro AsnArg Ser Thr Lys Gly Gly Gln Gln Lys Asn Ser Gln 500 505 510 Lys Gly GlnHis Ile Glu Gly Arg Gln Thr Gln Ser Arg Pro Ile Gln 515 520 525 Asn ValPro Gly Pro His Arg Thr Ile His His Ala Ser Ala Pro Leu 530 535 540 ThrAsp Asn Asp Arg Arg Asn Glu Pro Ser Gly Ser Thr Ser Pro Arg 545 550 555560 Met Leu Thr Pro Ile Asn Glu Glu Ala Asp Pro Leu Asp Asp Ala Asp 565570 575 Asp Glu Thr Ser Ser Leu Pro Pro Leu Glu Ser Asp Asp Glu Glu Gln580 585 590 Asp Arg Asp Gly Thr Ser Asn Arg Thr Pro Thr Val Ala Pro ProAla 595 600 605 Pro Val Tyr Arg Asp His Ser Glu Lys Lys Glu Leu Pro GlnAsp Glu 610 615 620 Gln Gln Asp Gln Asp His Thr Gln Glu Ala Arg Asn GlnAsp Ser Asp 625 630 635 640 Asn Thr Gln Ser Glu His Ser Phe Glu Glu MetTyr Arg His Ile Leu 645 650 655 Arg Ser Gln Gly Pro Phe Asp Ala Val LeuTyr Tyr His Met Met Lys 660 665 670 Asp Glu Pro Val Val Phe Ser Thr SerAsp Gly Lys Glu Tyr Thr Tyr 675 680 685 Pro Asp Ser Leu Glu Glu Glu TyrPro Pro Trp Leu Thr Glu Lys Glu 690 695 700 Ala Met Asn Glu Glu Asn ArgPhe Val Thr Leu Asp Gly Gln Gln Phe 705 710 715 720 Tyr Trp Pro Val MetAsn His Lys Asn Lys Phe Met Ala Ile Leu Gln 725 730 735 His His Gln 19251 PRT Ebola zaire 19 Met Ala Lys Ala Thr Gly Arg Tyr Asn Leu Ile SerPro Lys Lys Asp 1 5 10 15 Leu Glu Lys Gly Val Val Leu Ser Asp Leu CysAsn Phe Leu Val Ser 20 25 30 Gln Thr Ile Gln Gly Trp Lys Val Tyr Trp AlaGly Ile Glu Phe Asp 35 40 45 Val Thr His Lys Gly Met Ala Leu Leu His ArgLeu Lys Thr Asn Asp 50 55 60 Phe Ala Pro Ala Trp Ser Met Thr Arg Asn LeuPhe Pro His Leu Phe 65 70 75 80 Gln Asn Pro Asn Ser Thr Ile Glu Ser ProLeu Trp Ala Leu Arg Val 85 90 95 Ile Leu Ala Ala Gly Ile Gln Asp Gln LeuIle Asp Gln Ser Leu Ile 100 105 110 Glu Pro Leu Ala Gly Ala Leu Gly LeuIle Ser Asp Trp Leu Leu Thr 115 120 125 Thr Asn Thr Asn His Phe Asn MetArg Thr Gln Arg Val Lys Glu Gln 130 135 140 Leu Ser Leu Lys Met Leu SerLeu Ile Arg Ser Asn Ile Leu Lys Phe 145 150 155 160 Ile Asn Lys Leu AspAla Leu His Val Val Asn Tyr Asn Gly Leu Leu 165 170 175 Ser Ser Ile GluIle Gly Thr Gln Asn His Thr Ile Ile Ile Thr Arg 180 185 190 Thr Asn MetGly Phe Leu Val Glu Leu Gln Glu Pro Asp Lys Ser Ala 195 200 205 Met AsnArg Met Lys Pro Gly Pro Ala Lys Phe Ser Leu Leu His Glu 210 215 220 SerThr Leu Lys Ala Phe Thr Gln Gly Ser Ser Thr Arg Met Gln Ser 225 230 235240 Leu Ile Leu Glu Phe Asn Ser Ser Leu Ala Ile 245 250 20 324 PRT Ebolazaire 20 Met Glu Ala Ser Tyr Glu Arg Gly Arg Pro Arg Ala Ala Arg Gln His1 5 10 15 Ser Arg Asp Gly His Asp His His Val Arg Ala Arg Ser Ser SerArg 20 25 30 Glu Asn Tyr Arg Gly Glu Tyr Arg Gln Ser Arg Ser Ala Ser GlnVal 35 40 45 Arg Val Pro Thr Val Phe His Lys Lys Arg Val Glu Pro Leu ThrVal 50 55 60 Pro Pro Ala Pro Lys Asp Ile Cys Pro Thr Leu Lys Lys Gly PheLeu 65 70 75 80 Cys Asp Ser Ser Phe Cys Lys Lys Asp His Gln Leu Glu SerLeu Thr 85 90 95 Asp Arg Glu Leu Leu Leu Leu Ile Ala Arg Lys Thr Cys GlySer Val 100 105 110 Glu Gln Gln Leu Asn Ile Thr Ala Pro Lys Asp Ser ArgLeu Ala Asn 115 120 125 Pro Thr Ala Asp Asp Phe Gln Gln Glu Glu Gly ProLys Ile Thr Leu 130 135 140 Leu Thr Leu Ile Lys Thr Ala Glu His Trp AlaArg Gln Asp Ile Arg 145 150 155 160 Thr Ile Glu Asp Ser Lys Leu Arg AlaLeu Leu Thr Leu Cys Ala Val 165 170 175 Met Thr Arg Lys Phe Ser Lys SerGln Leu Ser Leu Leu Cys Glu Thr 180 185 190 His Leu Arg Arg Glu Gly LeuGly Gln Asp Gln Ala Glu Pro Val Leu 195 200 205 Glu Val Tyr Gln Arg LeuHis Ser Asp Lys Gly Gly Ser Phe Glu Ala 210 215 220 Ala Leu Trp Gln GlnTrp Asp Leu Gln Ser Leu Ile Met Phe Ile Thr 225 230 235 240 Ala Phe LeuAsn Ile Ala Leu Gln Leu Pro Cys Glu Ser Ser Ala Val 245 250 255 Val ValSer Gly Leu Arg Thr Leu Val Pro Gln Ser Asp Asn Glu Glu 260 265 270 AlaSer Thr Asn Pro Gly Thr Cys Ser Trp Ser Asp Glu Gly Thr Ser 275 280 285Ile Gln Gln Gln Leu Ala Ser Cys Leu His Arg Thr Arg Gly Asp Trp 290 295300 His Ala Ala Leu Lys Phe Leu Phe Tyr Phe Ser Phe Leu Phe Arg Ile 305310 315 320 Gly Phe Cys Phe 21 340 PRT Ebola zaire 21 Met Thr Thr ArgThr Lys Gly Arg Gly His Thr Ala Ala Thr Thr Gln 1 5 10 15 Asn Asp ArgMet Pro Gly Pro Glu Leu Ser Gly Trp Ile Ser Glu Gln 20 25 30 Leu Met ThrGly Arg Ile Pro Val Ser Asp Ile Phe Cys Asp Ile Glu 35 40 45 Asn Asn ProGly Leu Cys Tyr Ala Ser Gln Met Gln Gln Thr Lys Pro 50 55 60 Asn Pro LysThr Arg Asn Ser Gln Thr Gln Thr Asp Pro Ile Cys Asn 65 70 75 80 His SerPhe Glu Glu Val Val Gln Thr Leu Ala Ser Leu Ala Thr Val 85 90 95 Val GlnGln Gln Thr Ile Ala Ser Glu Ser Leu Glu Gln Arg Ile Thr 100 105 110 SerLeu Glu Asn Gly Leu Lys Pro Val Tyr Asp Met Ala Lys Thr Ile 115 120 125Ser Ser Leu Asn Arg Val Cys Ala Glu Met Val Ala Lys Tyr Asp Leu 130 135140 Leu Val Met Thr Thr Gly Arg Ala Thr Ala Thr Ala Ala Ala Thr Glu 145150 155 160 Ala Tyr Trp Ala Glu His Gly Gln Pro Pro Pro Gly Pro Ser LeuTyr 165 170 175 Glu Glu Ser Ala Ile Arg Gly Lys Ile Glu Ser Arg Asp GluThr Val 180 185 190 Pro Gln Ser Val Arg Glu Ala Phe Asn Asn Leu Asn SerThr Thr Ser 195 200 205 Leu Thr Glu Glu Asn Phe Gly Lys Pro Asp Ile SerAla Lys Asp Leu 210 215 220 Arg Asn Ile Met Tyr Asp His Leu Pro Gly PheGly Thr Ala Phe His 225 230 235 240 Gln Leu Val Gln Val Ile Cys Lys LeuGly Lys Asp Ser Asn Ser Leu 245 250 255 Asp Ile Ile His Ala Glu Phe GlnAla Ser Leu Ala Glu Gly Asp Ser 260 265 270 Pro Gln Cys Ala Leu Ile GlnIle Thr Lys Arg Val Pro Ile Phe Gln 275 280 285 Asp Ala Ala Pro Pro ValIle His Ile Arg Ser Arg Gly Asp Ile Pro 290 295 300 Arg Ala Cys Gln LysSer Leu Arg Pro Val Pro Pro Ser Pro Lys Ile 305 310 315 320 Asp Arg GlyTrp Val Cys Val Phe Gln Leu Gln Asp Gly Lys Thr Leu 325 330 335 Gly LeuLys Ile 340 22 326 PRT Ebola zaire 22 Met Arg Arg Val Ile Leu Pro ThrAla Pro Pro Glu Tyr Met Glu Ala 1 5 10 15 Ile Tyr Pro Val Arg Ser AsnSer Thr Ile Ala Arg Gly Gly Asn Ser 20 25 30 Asn Thr Gly Phe Leu Thr ProGlu Ser Val Asn Gly Asp Thr Pro Ser 35 40 45 Asn Pro Leu Arg Pro Ile AlaAsp Asp Thr Ile Asp His Ala Ser His 50 55 60 Thr Pro Gly Ser Val Ser SerAla Phe Ile Leu Glu Ala Met Val Asn 65 70 75 80 Val Ile Ser Gly Pro LysVal Leu Met Lys Gln Ile Pro Ile Trp Leu 85 90 95 Pro Leu Gly Val Ala AspGln Lys Thr Tyr Ser Phe Asp Ser Thr Thr 100 105 110 Ala Ala Ile Met LeuAla Ser Tyr Thr Ile Thr His Phe Gly Lys Ala 115 120 125 Thr Asn Pro LeuVal Arg Val Asn Arg Leu Gly Pro Gly Ile Pro Asp 130 135 140 His Pro LeuArg Leu Leu Arg Ile Gly Asn Gln Ala Phe Leu Gln Glu 145 150 155 160 PheVal Leu Pro Pro Val Gln Leu Pro Gln Tyr Phe Thr Phe Asp Leu 165 170 175Thr Ala Leu Lys Leu Ile Thr Gln Pro Leu Pro Ala Ala Thr Trp Thr 180 185190 Asp Asp Thr Pro Thr Gly Ser Asn Gly Ala Leu Arg Pro Gly Ile Ser 195200 205 Phe His Pro Lys Leu Arg Pro Ile Leu Leu Pro Asn Lys Ser Gly Lys210 215 220 Lys Gly Asn Ser Ala Asp Leu Thr Ser Pro Glu Lys Ile Gln AlaIle 225 230 235 240 Met Thr Ser Leu Gln Asp Phe Lys Ile Val Pro Ile AspPro Thr Lys 245 250 255 Asn Ile Met Gly Ile Glu Val Pro Glu Thr Leu ValHis Lys Leu Thr 260 265 270 Gly Lys Lys Val Thr Ser Lys Asn Gly Gln ProIle Ile Pro Val Leu 275 280 285 Leu Pro Lys Tyr Ile Gly Leu Asp Pro ValAla Pro Gly Asp Leu Thr 290 295 300 Met Val Ile Thr Gln Asp Cys Asp ThrCys His Ser Pro Ala Ser Leu 305 310 315 320 Pro Ala Val Ile Glu Lys 32523 288 PRT Ebola zaire 23 Met Glu Ala Ser Tyr Glu Arg Gly Arg Pro ArgAla Ala Arg Gln His 1 5 10 15 Ser Arg Asp Gly His Asp His His Val ArgAla Arg Ser Ser Ser Arg 20 25 30 Glu Asn Tyr Arg Gly Glu Tyr Arg Gln SerArg Ser Ala Ser Gln Val 35 40 45 Arg Val Pro Thr Val Phe His Lys Lys ArgVal Glu Pro Leu Thr Val 50 55 60 Pro Pro Ala Pro Lys Asp Ile Cys Pro ThrLeu Lys Lys Gly Phe Leu 65 70 75 80 Cys Asp Ser Ser Phe Cys Lys Lys AspHis Gln Leu Glu Ser Leu Thr 85 90 95 Asp Arg Glu Leu Leu Leu Leu Ile AlaArg Lys Thr Cys Gly Ser Val 100 105 110 Glu Gln Gln Leu Asn Ile Thr AlaPro Lys Asp Ser Arg Leu Ala Asn 115 120 125 Pro Thr Ala Asp Asp Phe GlnGln Glu Glu Gly Pro Lys Ile Thr Leu 130 135 140 Leu Thr Leu Ile Lys ThrAla Glu His Trp Ala Arg Gln Asp Ile Arg 145 150 155 160 Thr Ile Glu AspSer Lys Leu Arg Ala Leu Leu Thr Leu Cys Ala Val 165 170 175 Met Thr ArgLys Phe Ser Lys Ser Gln Leu Ser Leu Leu Cys Glu Thr 180 185 190 His LeuArg Arg Glu Gly Leu Gly Gln Asp Gln Ala Glu Pro Val Leu 195 200 205 GluVal Tyr Gln Arg Leu His Ser Asp Lys Gly Gly Ser Phe Glu Ala 210 215 220Ala Leu Trp Gln Gln Trp Asp Arg Gln Ser Leu Ile Met Phe Ile Thr 225 230235 240 Ala Phe Leu Asn Ile Ala Leu Gln Leu Pro Cys Glu Ser Ser Ala Val245 250 255 Val Val Ser Gly Leu Arg Thr Leu Val Pro Gln Ser Asp Asn GluGlu 260 265 270 Ala Ser Thr Asn Pro Gly Thr Cys Ser Trp Ser Asp Glu GlyThr Pro 275 280 285 24 11 PRT Ebola zaire 24 Val Tyr Gln Val Asn Asn LeuGlu Glu Ile Cys 1 5 10 25 23 PRT Ebola zaire 25 Leu Lys Phe Ile Asn LysLeu Asp Ala Leu Leu Val Val Asn Tyr Asn 1 5 10 15 Gly Leu Leu Ser SerIle Phe 20 26 8 PRT Ebola zaire 26 Gly Gln Phe Leu Phe Ala Ser Leu 1 527 9 PRT Ebola zaire 27 Asp Ala Val Leu Tyr Tyr His Met Met 1 5 28 9 PRTEbola zaire 28 Ser Phe Lys Ala Ala Leu Ser Ser Leu 1 5 29 15 PRT Ebolazaire 29 Trp Ile Pro Tyr Phe Gly Pro Ala Ala Glu Gly Ile Tyr Thr Glu 1 510 15 30 14 PRT Ebola zaire 30 Asn Tyr Asn Gly Leu Leu Ser Ser Ile GluGly Thr Gln Asn 1 5 10 31 25 PRT Ebola zaire 31 Arg Met Lys Pro Gly ProAla Lys Phe Ser Leu Leu His Glu Ser Thr 1 5 10 15 Leu Lys Ala Phe ThrGln Gly Ser Ser 20 25 32 15 PRT Ebola zaire 32 Phe Ser Lys Ser Gln LeuSer Leu Leu Cys Glu Thr His Leu Arg 1 5 10 15 33 15 PRT Ebola zaire 33Asp Leu Gln Ser Leu Ile Met Phe Ile Thr Ala Phe Leu Asn Ile 1 5 10 15 348 PRT Ebola zaire 34 Arg Asn Ile Met Tyr Asp His Leu 1 5 35 8 PRT Ebolazaire 35 Met Val Ala Lys Tyr Asp Leu Leu 1 5 36 9 PRT Ebola zaire 36 CysAsp Ile Glu Asn Asn Pro Gly Leu 1 5 37 15 PRT Ebola zaire 37 Ala Phe LeuGln Glu Phe Val Leu Pro Pro Val Gln Leu Pro Gln 1 5 10 15 38 20 PRTEbola zaire 38 Phe Val Leu Pro Pro Val Gln Leu Pro Gln Tyr Phe Thr PheAsp Leu 1 5 10 15 Thr Ala Leu Lys 20 39 30 PRT Ebola zaire 39 Lys SerGly Lys Lys Gly Asn Ser Ala Asp Leu Thr Ser Pro Glu Lys 1 5 10 15 IleGln Ala Ile Met Thr Ser Leu Gln Asp Phe Lys Ile Val 20 25 30 40 15 PRTEbola zaire 40 Pro Leu Arg Leu Leu Arg Ile Gly Asn Gln Ala Phe Leu GlnGlu 1 5 10 15 41 15 PRT Ebola zaire 41 Arg Ile Gly Asn Gln Ala Phe LeuGln Glu Phe Val Leu Pro Pro 1 5 10 15 42 9 PRT Ebola zaire 42 Tyr PheGly Pro Ala Ala Glu Gly Ile 1 5 43 9 PRT Ebola zaire 43 Lys Phe Ile AsnLys Leu Asp Ala Leu 1 5 44 9 PRT Ebola zaire 44 Asn Tyr Asn Gly Leu LeuSer Ser Ile 1 5 45 9 PRT Ebola zaire 45 Pro Gly Pro Ala Lys Phe Ser LeuLeu 1 5 46 10 PRT Ebola zaire 46 Leu Ser Leu Leu Cys Glu Thr His Leu Arg1 5 10 47 9 PRT Ebola zaire 47 Met Phe Ile Thr Ala Phe Leu Asn Ile 1 548 9 PRT Ebola zaire 48 Glu Phe Val Leu Pro Pro Val Gln Leu 1 5 49 6 PRTEbola zaire 49 Phe Leu Val Pro Pro Val 1 5 50 11 PRT Ebola zaire 50 GlnTyr Phe Thr Phe Asp Leu Thr Ala Leu Lys 1 5 10 51 9 PRT Ebola zaire 51Thr Ser Pro Glu Lys Ile Gln Ala Ile 1 5 52 8 PRT Ebola zaire 52 Arg IleGly Asn Gln Ala Phe Leu 1 5 53 8 PRT Ebola zaire 53 Gln Ala Phe Leu GlnGlu Phe Val 1 5

What is claimed is:
 1. An isolated GP Ebola peptide comprising thesequence specified in SEQ ID NO:29, or a peptide fragment comprising atleast 9 consecutive amino acids.
 2. The peptide of claim 1, wherein thepeptide fragment has the sequence of YFGPAAEGI (SEQ ID NO:42).
 3. Anisolated NP Ebola peptide comprising the sequence specified in SEQ IDNO:24.
 4. An isolated NP Ebola peptide comprising the sequence specifiedin SEQ ID NO:26, SEQ ID NO:27 or SEQ ID NO:28.
 5. An isolated VP24 Ebolapeptide comprising the sequence specified in SEQ ID NO:25, or a peptidefragment comprising at least 9 consecutive amino acids.
 6. The peptideof claim 5, wherein the peptide fragment has the sequence of KFINKLDAL(SEQ ID NO:43).
 7. An isolated VP24 Ebola peptide comprising thesequence specified in SEQ ID NO:30, or a peptide fragment comprising atleast 9 consecutive amino acids.
 8. The peptide of claim 7, wherein thepeptide fragment has the sequence of NYNGLLSSI (SEQ ID NO:44).
 9. Anisolated VP24 Ebola peptide comprising the sequence specified in SEQ IDNO:31, or a peptide fragment comprising at least 9 consecutive aminoacids.
 10. The peptide of claim 9, wherein the peptide fragment has thesequence of PGPAKFSLL (SEQ ID NO:45).
 11. An isolated VP30 Ebola peptidecomprising the sequence specified in SEQ ID NO:32, or a peptide fragmentcomprising at least 9 consecutive amino acids.
 12. The peptide of claim11, wherein the peptide fragment has the sequence of LSLLCETHLR (SEQ IDNO:46).
 13. An isolated VP30 Ebola peptide comprising the sequencespecified in SEQ ID NO:33, or a peptide fragment comprising at least 9consecutive amino acids.
 14. The peptide of claim 13, wherein thepeptide fragment has the sequence of MFITAFLNI (SEQ ID NO:47).
 15. Anisolated VP35 Ebola peptide comprising the sequence specified in SEQ IDNO:34, SEQ ID NO:35 or SEQ ID NO:36.
 16. An isolated VP40 Ebola peptidecomprising the sequence specified in SEQ ID NO:37, or a peptide fragmentcomprising at least 9 consecutive amino acids.
 17. The peptide of claim16, wherein the peptide fragment has the sequence of EFVLPPVQL (SEQ IDNO:48).
 18. An isolated VP40 Ebola peptide comprising the sequencespecified in SEQ ID NO:38, or a peptide fragment comprising at least 6consecutive amino acids.
 19. The peptide of claim 18, wherein thepeptide fragment has the sequence of FLVPPV (SEQ ID NO:49) orQYFTFDLTALK (SEQ ID NO:50).
 20. An isolated VP40 Ebola peptidecomprising the sequence specified in SEQ ID NO:39, or a peptide fragmentcomprising at least 9 consecutive amino acids.
 21. The peptide of claim20, wherein the peptide fragment has the sequence of TSPEKIQAI (SEQ IDNO:51).
 22. An isolated VP40 Ebola peptide comprising the sequencespecified in SEQ ID NO:40, or a peptide fragment comprising at least 8consecutive amino acids.
 23. The peptide of claim 22, wherein thepeptide fragment has the sequence of RIGNQAFL (SEQ ID NO:52).
 24. Anisolated VP40 Ebola peptide comprising the sequence specified in SEQ IDNO:41, or a peptide fragment comprising at least 8 consecutive aminoacids.
 25. The peptide of claim 24, wherein the peptide fragment has thesequence of QAFLQEFV (SEQ ID NO:53).
 26. An isolated DNA fragment whichencodes the GP Ebola peptide of claim
 1. 27. A DNA fragment whichencodes the NP Ebola peptide of claim3.
 28. A DNA fragment which encodesthe SEQ ID NO:26 NP Ebola peptide of claim
 4. 29. A DNA fragment whichencodes the SEQ ID NO:27 NP Ebola peptide of claim
 4. 30. A DNA fragmentwhich encodes the SEQ ID NO:28 NP Ebola peptide of claim
 4. 31. A DNAfragment which encodes the VP24 Ebola peptide of claim
 5. 32. A DNAfragment which encodes the VP24 Ebola peptide of claim
 7. 33. A DNAfragment which encodes the VP24 Ebola peptide of claim
 9. 34. A DNAfragment which encodes the VP30 Ebola peptide of claim
 11. 35. A DNAfragment which encodes the VP30 Ebola peptide of claim
 13. 36. A DNAfragment which encodes the SEQ ID NO:34 VP35 Ebola peptide of claim 15.37. A DNA fragment which encodes the SEQ ID NO:35 VP35 Ebola peptide ofclaim
 15. 38. A DNA fragment which encodes the SEQ ID NO:36 VP35 Ebolapeptide of claim
 15. 39. A DNA fragment which encodes the VP40 Ebolapeptide of claim
 16. 40. A DNA fragment which encodes the VP40 Ebolapeptide of claim
 18. 41. A DNA fragment which encodes the VP40 Ebolapeptide of claim
 20. 42. A DNA fragment which encodes the VP40 Ebolapeptide of claim
 22. 43. A DNA fragment which encodes the VP40 Ebolapeptide of claim
 24. 44. A recombinant DNA construct comprising: (i) avector, and (ii) at least one of the Ebola virus DNA fragments encodinga peptide selected from the group consisting of SEQ ID NOs: 24, 25, 26,27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44,45, 46, 47, 48, 49, 50, 51, 52 and
 53. 45. The recombinant DNA constructof claim 44, wherein the DNA fragment encodes the peptide of SEQ IDNO:24 or SEQ ID NO:25.
 46. The recombinant DNA construct of claim 44wherein said DNA fragment induces a cytotoxic T lymphocyte response. 47.The recombinant DNA construct according to claim 44 wherein said vectoris an expression vector.
 48. The recombinant DNA construct according toclaim 44 wherein said vector is a VEE virus replicon vector.
 49. Therecombinant DNA construct according to claim 44 wherein said vector is aeukaryotic vector.
 50. The recombinant DNA construct of claim 44 whereinsaid vector is selected from the group consisting of Venezuelan EquineEncephalitis (VEE) virus replicon vector, eastern equine encephalitisvirus replicon vector, western equine encephalitis virus repliconvector, Semliki forest virus replicon vector and Sindbis virus repliconvector.
 51. A pharmaceutical composition comprising SEQ ID NO:24, SEQ IDNO:25, or both.
 52. A pharmaceutical composition comprising a peptideselected from the group consisting of SEQ ID NOs: 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,47, 48, 49, 50, 51, 52 and 53, and mixtures thereof, in an effectiveimmunogenic amount in a pharmaceutically acceptable carrier and/oradjuvant.
 53. A vaccine against Ebola infection comprising a peptideselected from the group consisting of SEQ ID NOs: 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,47, 48, 49, 50, 51, 52 and 53, and mixtures thereof, in an effectiveimmunogenic amount in a pharmaceutically acceptable carrier and/oradjuvant.
 54. A vaccine against Ebola infection comprising SEQ ID NO:24,SEQ ID NO:25, or both, in an effective immunogenic amount in apharmaceutically acceptable carrier and/or adjuvant.
 55. A vaccineagainst Ebola infection comprising virus replicon particles expressingat least one of the peptides specified by SEQ ID NOs:24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,47, 48, 49, 50, 51, 52 and 53, in an effective immunogenic amount in apharmaceutically acceptable carrier and/or adjuvant.
 56. The vaccine ofclaim 55, wherein the virus replicon particles are produced from areplicon vector selected from the group consisting of Venezuelan EquineEncephalitis (VEE) virus, eastern equine encephalitis, western equineencephalitis, Semliki forest and Sindbis.
 57. A vaccine against Ebolainfection comprising virus replicon particles expressing at least one ofthe peptides specified by SEQ ID NOs:24 and 25, in an effectiveimmunogenic amount in a pharmaceutically acceptable carrier and/oradjuvant.
 58. The vaccine of claim 57, wherein the virus repliconparticles are produced from a replicon vector selected from the groupconsisting of Venezuelan Equine Encephalitis (VEE) virus, eastern equineencephalitis, western equine encephalitis, Semliki forest and Sindbis.59. A method for inducing in a mammal a cytotoxic T lymphocyte responseto an Ebola peptide comprising the step of: administering to a mammal animmunogenic composition comprising a peptide selected from the groupconsisting of SEQ ID NOs: 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52and 53, and mixtures thereof, in an effective immunogenic amount in apharmaceutically acceptable carrier and/or adjuvant, under suchconditions that the peptide induces a protective cytotoxic T lymphocyteresponse.
 60. A method for inducing in a mammal a cytotoxic T lymphocyteresponse to an Ebola peptide comprising the step of: administering to amammal an immunogenic composition comprising a peptide selected from thegroup consisting of SEQ ID NOs:24 and 25, and mixtures thereof, in aneffective immunogenic amount in a pharmaceutically acceptable carrierand/or adjuvant, under such conditions that the peptide induces aprotective cytotoxic T lymphocyte response.
 61. A method for inducing ina mammal a cytotoxic T lymphocyte response to an Ebola peptidecomprising the step of: administering to a mammal a recombinant DNAconstruct that expresses a peptide selected from the group consisting ofSEQ ID NOs: 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52 and 53, andmixtures thereof, in an effective immunogenic amount in apharmaceutically acceptable carrier and/or adjuvant, under suchconditions that the peptide induces a protective cytotoxic T lymphocyteresponse.
 62. The method of claim 61, wherein the recombinant DNAconstruct comprises: (i) a replicon vector selected from the groupconsisting of Venezuelan Equine Encephalitis (VEE) virus, eastern equineencephalitis, western equine encephalitis, Semliki forest and Sindbis,and (ii) at least one of the Ebola virus DNA fragments encoding apeptide selected from the group consisting of SEQ ID NOs: 24, 25, 26,27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44,45, 46, 47, 48, 49, 50, 51, 52 and 53, and mixtures thereof.
 63. Amethod for inducing in a mammal a cytotoxic T lymphocyte response to anEbola peptide comprising the step of: administering to a mammal arecombinant DNA construct that expresses a peptide selected from thegroup consisting of SEQ ID NOs:24 and 25, and mixtures thereof, in aneffective immunogenic amount in a pharmaceutically acceptable carrierand/or adjuvant, under such conditions that the peptide induces aprotective cytotoxic T lymphocyte response.
 64. The method of claim 63,wherein the recombinant DNA construct comprises: (i) a replicon vectorselected from the group consisting of Venezuelan Equine Encephalitis(VEE) virus, eastern equine encephalitis, western equine encephalitis,Semliki forest and Sindbis, and (ii) at least one of the Ebola virus DNAfragments encoding a peptide selected from the group consisting of SEQID NOs: 24 and 25, and mixtures thereof.
 65. An immunogenic compositioncomprising Ebola peptides VP30, VP35, and VP40.
 66. The immunogeniccomposition of claim 65 wherein the VP30 peptide has the amino acidsequence of SEQ ID NO:20.
 67. The immunogenic composition of claim 65wherein the VP35 peptide has the amino acid sequence of SEQ ID NO:21 68.The immunogenic composition of claim 65 wherein the VP40 peptide has theamino acid sequence of SEQ ID NO:22.
 69. The immunogenic composition ofclaim 65 wherein the VP30 peptide has the amino acid sequence of SEQ IDNO:20, the VP35 peptide has the amino acid sequence of SEQ ID NO:21, andthe VP40 peptide has the amino acid sequence of SEQ ID NO:22.